Anti-avbeta8 antibodies and compositions and uses thereof

ABSTRACT

The invention provides antibodies, and antigen-binding fragments thereof, that specifically bind to αvβ8 integrin. The invention includes uses, and associated methods of using the antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/561,530, filed Sep. 5, 2019, which claims priority to U.S. Ser. No.62/890,945 filed Aug. 23, 2019, and U.S. Ser. No. 62/728,688 filed Sep.7, 2018, the contents of each of which are incorporated herein byreference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as38972A_SequenceListing.txt of 180 KB, created on May 5, 2022 andsubmitted in the United States Patent and Trademark Office via EFS-Web,is incorporated herein by reference.

PARTIES TO A JOINT RESEARCH STATEMENT

The presently claimed invention was made by or on behalf of the belowlisted parties to a joint research agreement. The joint researchagreement was in effect on or before the date the claimed invention wasmade and the claimed invention was made as a result of activitiesundertaken within the scope of the joint research agreement. The partiesto the joint research agreement are THE REGENTS OF THE UNIVERSITY OFCALIFORNIA on behalf of its SAN FRANCISCO CAMPUS and PFIZER INC.

FIELD

The present invention relates to antibodies, and antigen-bindingfragments thereof, that specifically bind αvβ8 integrin, andcompositions, methods and uses thereof.

BACKGROUND

Transforming growth factor β (TGFβ) is a potent suppressor of adaptiveand innate immunity and an important mediator of immune suppression by asubset of regulatory T cells. TGFβ is required for the induction of Th17cells, which can promote tumor progression through induction ofgranulocytic inflammation and promotes epithelial to mesenchymaltransformation of tumor cells and secretion and accumulation of afibrotic tumor stroma that may contribute to exclusion of immune cellsfrom some solid tumors. For all of these reasons, inhibition of TGFβ hasbeen explored as an adjunctive immunotherapy, especially in so-called“immune-excluded” tumors (Gorelik et al. Nat. Med. 7:1118-1122, 2001;Tauriello et al. Nature 554:538-543, 2018; Mariathasan et al. Nature554:544-548, 2018, Dodagatta et al. J Immunother Cancer. 7: 62. 2019;U.S. Pat. No. 10,167,334). However, since TGFβ plays importanthomeostatic roles in many biological systems, systemic targeting of TGFβsignaling presents numerous challenges due to unwanted side effects(Hata and Akhurst Nat. Rev. Drug Dev. 11, 791-811, 2012; Akhurst et al.Cold Spring Harbor Perspectives. 10, 2017; Flavell et al. Nat. Rev.Immunol. 10:554-567, 2010).

Previous studies have shown that inhibition of TGFβ signaling canenhance responses to radiation or vaccine therapies in combination withcheckpoint inhibition (Vanpouille-Box et al. Cancer Res. 75:2232-2242,2015; Terabe et al. OncoImmunology 6(5):e1308616, 2017). In vivoactivity of TGFβ is regulated via several mechanisms. For example, TGFβis secreted as an inactive or latent complex, where the cleaved latencyassociated peptide (LAP) domain encases the active TGFβ mature peptide.Latent-TGFβ can be covalently linked to the extracellular matrix throughlatent TGFβ binding protein (LTBP) or displayed on the cell surface byGlycoprotein-A Repetitions Predominant protein (GARP). Early in vitrodata showed that the latent complex of TGFβ can be activated by hightemperature, acidic pH, and various proteases (Annes et al. J Cell Sci.116:217-24, 2003), however the importance of these mechanisms in vivoremains to be determined.

A role for members of the αv Integrin family, specifically αvβ1, αvβ6,and αvβ8, has been demonstrated for latent-TGFβ activation. Integrinαvβ8 is a transmembrane noncovalent heterodimer consisting of ITGαV andITGβ8 subunits. αvβ8 expression is unique among αv integrins, where itsexpression by immune cells such as dendritic cells, T regulatory cells,and tumor associated macrophages has emerged as a contextual activatorof TGFβ for regulation of active immune responses. αvβ8 expression bydendritic cells (DCs) acts as a mediator of TGFβ production duringT-cell stimulation and strongly influences the differentiation anddevelopment of Tregs and Th17 cells at the expense of Th1differentiation during immune responses. Mice with conditional deletionof Itgb8 in DCs or all leukocytes demonstrate a dramatic inhibition ofTGFβ-dependent induction of antigen-specific Th17 cells and aresubsequently protected from organ dysfunction in certain preclinicalmodels of autoimmunity, such as multiple sclerosis (experimentalauto-immune encephalomyelitis) and allergic asthma (Travis et al.Nature. 449(7160):361-5, 2007; Melton et al. J Clin. Invest.120(12):4436-44, 2010).

TGFβ plays a role in both the differentiation and recruitment of immunesuppressor cells to the tumor, and as a tumor intrinsic factor thatcontributes to an immune suppressive tumor microenvironment. In somecancers, TGFβ can be tumor-promoting by influencing numerous aspects ofthe tumor microenvironment including angiogenesis, metastasis,epithelial-mesenchymal transition, and perhaps most importantly,suppression of infiltrating immune cells.

Accordingly, in view of the prominent role of TGFβ in the tumormicroenvironment and the numerous challenges associated with thesystemic targeting of TGFβ signaling (Hata and Akhurst Nat. Rev. DrugDev. 11, 791-811, 2012; Akhurst et al. Cold Spring Harbor Perspectives10, 2017; Flavell et al. Nat. Rev. Immunol. 10:554-567, 2010), the needexists for developing strategies for the selective inhibition ofαvβ8-dependent latent-TGFβ activation.

SUMMARY OF THE INVENTION

Disclosed herein are antibodies (e.g., humanized and chimericantibodies), and antigen-binding fragments thereof, that specificallybind to αvβ8 integrin (also interchangeably referred to herein as“AVB8”, “αvβ8” or “αvb8”) (e.g., αvβ8 integrin from human, mouse,cynomolgus monkey, and/or rat). In certain aspects, antibodies andantigen-binding fragments thereof bind to αvβ8 integrin, and ultimatelyreduce TGFβ (e.g., TGFβ1 and TGFβ3) signaling, e.g., in the tumor ortumor microenvironment.

Mature TGFβ is present in inactive or latent form in a complex with thelatency associated peptide (LAP) domain. Binding of αvβ8 integrin to LAPresults in release of active TGFβ (e.g., TGFβ1 and TGFβ3). Reducingbinding of αvβ8 integrin to LAP can prevent the release of active TGFβ,thereby reducing TGFβ signaling. TGFβ is known to have immunesuppressive effects, e.g., in the tumor microenvironment, thus reductionof TGFβ activity and/or signaling using the antibodies described hereincan result in activation of an immune response, e.g., an anti-tumorresponse in vivo.

Because of the restricted expression of αvβ8 integrin on immune cells(e.g., dendritic cells, T regulatory cells, tumor-associatedmacrophages) and tumor cells, antibodies disclosed herein can result ina more targeted, non-systemic reduction of TGFβ signaling. Thus,antibodies, and antigen binding fragments thereof, of the disclosureenable a more selective antagonism of TGFβ activity in the immune systemand/or the tumor microenvironment, thereby enhancing an anti-tumorimmune response in a subject. In some embodiments disclosed herein,antibodies against αvβ8 integrin have been shown to cause growthsuppression and/or complete tumor regression in animal models forseveral cancers, including, for example, squamous cell carcinoma, breastcancer, and/or colon cancer, alone or in combination with otherimmunomodulators, such as modulators of checkpoint inhibitors, (e.g.,inhibitors of PD-1, PD-L1, CTLA-4 or agonists of 4-1BB), or anti-cancertherapies, e.g., radiotherapy.

Accordingly, in certain aspects, the disclosure provides antibodies, andantigen-binding fragments thereof, that bind to αvβ8 integrin with highaffinity and specificity, nucleic acid molecules encoding antibodies andantigen-binding fragments thereof, expression vectors, host cells andmethods for making the same. In certain aspects, antibodies, andantigen-binding fragments thereof, exhibit altered effector functions(e.g., have reduced antibody-dependent cell-mediated cytotoxicity (ADCC)activity and/or reduced complement dependent cytotoxicity (CDC)activity). In certain aspects, anti-αvβ8 integrin antibodies andantigen-binding fragments thereof exhibit enhanced binding affinity forαvβ8 integrin as compared to murine hybridoma antibodies, andantigen-binding fragments thereof, from which they are derived.Humanized anti-αvβ8 integrin antibodies and antigen-binding fragmentsthereof disclosed herein can be used alone, or in combination with otheragents or therapeutic modalities, (e.g., immunomodulators or anti-cancertherapies) to treat, prevent and/or diagnose disorders, such ascancerous disorders (e.g., solid and soft-tissue tumors). Thus,compositions and methods for detecting αvβ8 integrin, as well as methodsfor treating various disorders, including cancer, using anti-αvβ8integrin antibodies and antigen-binding fragments thereof are disclosed.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments (E).

E1. An isolated antibody, or antigen-binding fragment thereof, thatspecifically binds to αvβ8 integrin, wherein said antibody, orantigen-binding fragment thereof, has at least one of the followingproperties:

-   -   i. a binding affinity, expressed as KD, for human αvβ8 integrin        that is less than the KD for the murine antibody ADWA11 as        disclosed in U.S. Pat. No. 9,969,804, which is herein        incorporated by reference in its entirety, confirming the amino        acid sequences and as set forth in, e.g., SEQ ID NO: 20-33 and        71-76 of the present description, e.g., the ADWA11 antibodies of        the invention have a KD less than 536 pM (e.g., 1, 5, 10, 20,        30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 370,        400, 450, 500, 510, 520, 530, 531, 532, 533, 534, or 535 pM);    -   ii. a KD for human αvβ8 integrin that is less than or equal to        200 pM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,        60, 70, 80, 90, 100, 150, 180, 190 or 200 pM), e.g., for        purified human αvβ8 integrin;    -   iii. a KD for human αvβ8 integrin that is less than or equal to        100 pM for purified human αvβ8 integrin;    -   iv. a KD for mouse αvβ8 integrin that is less than the KD for        the murine antibody ADWA11, e.g., less than 489 pM (e.g., 1, 5,        10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,        350, 370, 400, 450, 460, 470, 480, 485, 486, 487, or 488 pM);    -   v. a KD for mouse αvβ8 integrin that is 70.8+/−19.9 pM for        purified mouse αvβ8 integrin;    -   vi. a KD for cynomolgus monkey αvβ8 integrin that is less than        the KD for the murine antibody ADWA11, e.g., less than 507 pM        (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200,        250, 300, 350, 370, 400, 450, 500, 501, 502, 503, 504, 505, or        506 pM);    -   vii. a KD for cynomolgus αvβ8 integrin that is less than or        equal to 100 pM for purified cynomolgus αvβ8 integrin;    -   viii. a KD for rat αvβ8 integrin that is about 160 pM;    -   ix. approximately equivalent affinity for at least two, three,        or all of human, cynomolgus, mouse, and rat αvβ8 integrin, e.g.,        with a KD that is less than 100 pM (e.g., 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98 pM),        e.g., as determined using a Biacore affinity assay;    -   x. an IC50 for inhibiting TGFβ transactivation that is less than        that of the murine antibody ADWA11, e.g., less than 183 pM        (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60,        70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 180,        181, or 182 pM);    -   xi. an IC50 for inhibiting TGFβ transactivation in U251 cells of        about 199+/−93.6 pM;    -   xii. an IC50 for inhibiting TGFβ transactivation that is about        100 pM to about 300 pM;    -   xiii. an EC50 for U251 cells of about 126+/−34 pM (e.g., about        50, 60, 70 80, 90, 100, 110, 115, 120, 121, 122, 123, 124, 125,        126, 127, 128, 129, 130, 140, 150, 160, 170, 180, or 190 pM);    -   xiv. an EC50 for U251 cells of about 256+/−115 pM (e.g., about        120, 140, 160, 180, 200, 220, 240, 260, 280, 290, 300, 320, 340,        360, 380, 400 pM);    -   xv. an EC50 for U251 cells of about 80 pM to about 400 pM;    -   xvi. an EC50 for C8-S cells of about 115 pM;    -   xvii. an EC50 for C8-S cells of about 145+/−23.7 pM;    -   xviii. an EC50 for C8-S cells of about 110 pM to about 180 pM;    -   xix. at least one predicted human pharmacokinetic (PK) parameter        chosen from:        -   a. a clearance from central compartment (CL) of about            0.12-0.15 mL/h/kg;        -   b. an inter-compartmental distribution clearance (CLF) of            about 0.15-0.51 mL/h/kg;        -   c. a volume of distribution for the central compartment (V1)            of about 36-39 mL/kg;        -   d. a volume of distribution for the peripheral compartment            (V2) of about 21-33 mL/kg; and/or        -   e. a terminal half-life (t_(1/2)) of about 12 days;        -   f. a terminal half-life (t_(1/2)) of about 15-17 days; or    -   xx. no detectable binding to human Fcγ receptors or C1q.        E2. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E1, wherein the KD for human αvβ8 integrin is less        than the KD for the murine antibody ADWA11, e.g., less than 536        pM (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,        200, 250, 300, 350, 370, 400, 450, 500, 510, 520, 530, 531, 532,        533, 534, or 535 pM).        E3. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E1 or E2, wherein the KD for human αvβ8 integrin        is less than or equal to 100 pM (e.g., 1, 2, 3, 4, 5, 6, 7, 8,        9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pM), e.g., for        purified human αvβ8 integrin.        E4. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the KD for mouse        αvβ8 integrin is less than the KD for the murine antibody        ADWA11, e.g., less than 489 pM (e.g., 1, 5, 10, 20, 30, 40, 50,        60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 370, 400, 450,        460, 470, 480, 485, 486, 487, or 488 pM).        E5. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the KD for mouse        αvβ8 integrin is about 70.8+/−19.9 pM.        E6. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the KD for        cynomolgus monkey αvβ8 integrin is less than the KD for the        murine antibody ADWA11, e.g., less than 507 pM (e.g., 1, 5, 10,        20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,        370, 400, 450, 500, 501, 502, 503, 504, 505, or 506 pM).        E7. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the KD for        cynomolgus monkey αvβ8 integrin is less than 100 pM.        E8. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the KD for rat αvβ8        integrin is about 160 pM.        E9. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the isolated        antibody, or antigen-binding fragment thereof, shows        approximately equivalent affinity for at least two, three, or        all of human, cynomolgus, mouse, and rat αvβ8 integrin, e.g.,        with a KD that is less than 100 pM (e.g., 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95 pM), e.g., as        determined using a Biacore affinity assay.        E10. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the isolated        antibody, or antigen-binding fragment thereof, shows        approximately equivalent affinity for at least two, three, or        all of human, cynomolgus, mouse, and rat αvβ8 integrin, e.g.,        with a KD that is less than 100 pM (e.g., 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 98 pM),        e.g., as determined using a Biacore affinity assay.        E11. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the IC50 for        inhibiting TGFβ transactivation is less than the murine antibody        ADWA11, e.g., less than 183 pM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,        150, 160, 170, 175, 180, 181, or 182 pM).        E12. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the IC50 for        inhibiting TGFβ transactivation in U251 cells is about        199+/−93.6 pM.        E13. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the IC50 for        inhibiting TGFβ transactivation is about 100 pM to about 300 pM.        E14. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the EC50 for U251        cells is about 126 pM with a standard deviation of plus or minus        34 pM.        E15. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the EC50 for U251        cells is about 256 pM with a standard deviation of plus or minus        115 pM.        E16. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the EC50 for U251        cells is about 100 pM to about 400 pM.        E17. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the EC50 for C8-S        cells is about 115 pM.        E18. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the EC50 for C8-S        cells is about 145+/−23.7 pM.        E19. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the EC50 for C8-S        cells is about 110 pM to about 180 pM.        E20. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, having at least one        predicted human pharmacokinetic (PK) parameter chosen from the        group consisting of:    -   (i) a clearance from central compartment (CL) of about 0.12-0.15        mL/h/kg;    -   (ii) an inter-compartmental distribution clearance (CLF) of        about 0.15-0.51 mL/h/kg;    -   (iii) a volume of distribution for the central compartment (V1)        of about 36-39 mL/kg;    -   (iv) a volume of distribution for the peripheral compartment        (V2) of about 21-33 mL/kg; and/or    -   (v) a terminal half-life (t_(1/2)) of about 12-17 days.        E21. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the isolated        antibody, or antigen-binding fragment thereof, shows no        detectable binding to a human Fcγ receptor or C1q.        E22. The isolated antibody, or antigen-binding fragment thereof,        of any one of the preceding embodiments, wherein the antibody,        or antigen-binding fragment thereof, further has at least one of        the following properties:    -   (i) binds specifically to αvβ8 integrin (e.g., αvβ8 integrin        from human, mouse, cynomolgus monkey, and/or rat);    -   (ii) reduce an interaction between αvβ8 integrin and Latency        Associated Peptide (LAP);    -   (iii) reduces TGF-β signaling;    -   (iv) effectively blocks the αvβ8 integrin-mediated TGFβ        activation with an IC50≤10 nM;    -   (v) has a comparable Kd (within 5-fold) towards a non-human        primate (NHP) orthologue;    -   (vi) selectivity binds human αvβ8 and does not detectably bind a        homologue of αvβ8 (e.g., αvβ1, αvβ3, αvβ5 and αvβ6);    -   (vii) causes growth suppression and/or complete tumor regression        in an animal model for a cancer, alone or in combination with an        immunomodulatory agent, e.g., a modulators of checkpoint        inhibitors, e.g., inhibitors of PD-1, PD-L1, CTLA-4, or an        agonist of a stimulatory molecule, e.g., 4-1BB;    -   (viii) causes growth suppression and/or complete tumor        regression in an animal model for a cancer in combination with        an anti-cancer therapy, e.g., radiotherapy;    -   (ix) shows at least 60% reduction in tumor growth in a syngeneic        tumor graft model, e.g., when administered at ≤10 mg/kg, alone        or in combination with an immunomodulatory agent (e.g., an        inhibitor of PD-1, PD-L1, or CTLA-4);    -   (x) increases an anti-tumor response in the presence of one or        more immunomodulators, e.g., an antagonist of a checkpoint        inhibitor, e.g., an antagonist of PD-1, PD-L1, or CTLA-4, or an        activator of an immune response, e.g., 4-1BB agonist, when        administered to a subject;    -   (xi) has an efficacy that is not dependent upon the expression        of αvβ8 integrin in a tumor model;    -   (xii) increases the abundance of CD8+ GzmB+ T cells in the tumor        microenvironment;    -   (xiii) shows a decrease, e.g., at least a >80% decrease, in        tumor growth when used in combination with an antagonist of a        checkpoint inhibitor (e.g., an anti-PD-1 or anti-PD-L1        antibody), e.g., in a syngeneic model of squamous cell        carcinoma, breast cancer, and/or colon cancer;    -   (xiv) shows a statistically significant improvement in overall        survival of a subject, as determined by a Kaplan-Meier analysis;    -   (xv) has a high degree of thermal stability;    -   (xvi) shows minimal aggregation at high concentration; and    -   (xvii) may show reproducible expression and purity in        large-scale manufacturing conditions.        E23. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   one, two or three CDRs from a heavy chain variable region (e.g.,        H1, H2 or H3), and/or one, two, or three CDRs from a light chain        variable region (e.g., L1, L2 or L3) selected from:    -   (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8        or 14,        -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9            or 15,        -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10            or 16,        -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11            or 17,        -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 12            or 18, and        -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 13            or 19, or    -   (ii) a CDR-H1 comprising at least one amino acid alteration, but        not more than two, three or four alterations (e.g., a        substitution, deletion, or insertion, e.g., conservative        substitution) relative to SEQ ID NO: 8 or 14,        -   a CDR-H2 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 9 or 15,        -   a CDR-H3 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 10 or 16,        -   a CDR-L1 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 11 or 17,        -   a CDR-L2 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 12 or 18, or        -   a CDR-L3 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 13 or 19, optionally            wherein:        -   any of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3 do            not comprise the amino acid sequence of any of:        -   (a) SEQ ID NOs: 22, 23, 24, 25, 26, and 27, respectively,        -   (b) SEQ ID NOs: 28, 29, 30, 31, 32, and 33, respectively,        -   (c) SEQ ID NOs: 22, 23, 24, 71, 72, and 73, respectively, or        -   (d) SEQ ID NOs: 28, 29, 30, 74, 75, and 76, respectively.

Alternatively, or in combination with any of the embodiments providedherein (e.g., E1-E23), the antibody, or antigen-binding fragmentthereof, has one or more of the following aspects, features, andembodiments.

E24. An isolated antibody, or antigen-binding fragment thereof, thatspecifically binds to human αvβ8 integrin, comprising:

-   -   one, two or three CDRs from a heavy chain variable region (e.g.,        H1, H2 or H3), and/or one, two, or three CDRs from a light chain        variable region (e.g., L1, L2 or L3) selected from:    -   (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8,        -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9,        -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO:            10,        -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO:            11,        -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO:            12, and        -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO:            13, or    -   (ii) a CDR-H1 comprising at least one amino acid alteration, but        not more than two, three or four alterations (e.g., a        substitution, deletion, or insertion, e.g., conservative        substitution) relative to SEQ ID NO: 8,        -   a CDR-H2 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 9,        -   a CDR-H3 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 10,        -   a CDR-L1 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 11,        -   a CDR-L2 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 12, or        -   a CDR-L3 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 13, optionally wherein:        -   any of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3 do            not comprise the amino acid sequence of any of:        -   (a) SEQ ID NOs: 22, 23, 24, 25, 26, and 27, respectively, or        -   (b) SEQ ID NOs: 22, 23, 24, 71, 72, and 73, respectively.            E25. An isolated antibody, or antigen-binding fragment            thereof, that specifically binds to human αvβ8 integrin,            comprising:    -   one, two, or three complementarity determining regions (CDRs)        from a heavy chain variable region (e.g., H1, H2 or H3), and/or        one, two, or three CDRs from a light chain variable region        (e.g., L1, L2 or L3) selected from:    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 12,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 13.        E26. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E24 or E25, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9, and    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10.        E27. The isolated antibody, or antigen-binding fragment thereof,        of any of embodiments E24-E26, comprising:    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 12,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 13.        E28. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 12,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 13.        E29. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three complementarity determining regions (CDRs) from a        heavy chain variable region (e.g., H1, H2 or H3), and/or one,        two, or three CDRs from a light chain variable region (e.g., L1,        L2 or L3):    -   a CDR-H1 comprising the amino acid sequence of DYYMN (SEQ ID NO:        8);    -   a CDR-H2 comprising the amino acid sequence of        WIDPDXiGNTIYX₂PKFQG (SEQ ID NO: 131), wherein X₁ can be any one        of: an amino acid, an amino acid other than N, a conservative        substitution of N, N, or Q; and X₂ can be any one of: an amino        acid, an amino acid other than D, a conservative substitution of        D, D, or E;    -   a CDR-H3 comprising the amino acid sequence of RLLMDY (SEQ ID        NO: 10);    -   a CDR-L1 comprising the amino acid sequence of RSTKSLX₃HFNGNTYLF        (SEQ ID NO: 132), wherein X₃ can be any one of: an amino acid,        an amino acid other than L, a conservative substitution of L, L,        or S;    -   a CDR-L2 comprising the amino acid sequence of YYMSX₄LAS (SEQ ID        NO: 133), wherein X₄ can be any one of: an amino acid, an amino        acid other than N, a conservative substitution of N, N, or S;        and/or    -   a CDR-L3 comprising the amino acid sequence of X₅QSLEYPFT (SEQ        ID NO: 134), wherein X₅ can be any one of: an amino acid, an        amino acid other than M, a conservative substitution of M, M, or        Q;    -   e.g., wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and        CDR-L3 do not comprise the amino acid sequences of SEQ ID NOs:        22, 23, 24, 25, 26, and 27, respectively, or SEQ ID NOs: 22, 23,        24, 71, 72, and 73, respectively.        E30. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E29, wherein X₁ is Q and X₂ is E.        E31. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E29 or E30, wherein X₃ is S.        E32. The isolated antibody, or antigen-binding fragment thereof,        of any of embodiments E29-E31, wherein X₄ is S.        E33. The isolated antibody, or antigen-binding fragment thereof,        of any of embodiments E29-E32, wherein X₅ is Q.        E34. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E29, wherein X₁ is Q, X₂ is E, X₃ is S, and X₅ is        Q.        E35. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E29, wherein X₁ is Q, X₂ is E, and X₃ is S.        E36. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E29, wherein X₁ is Q, X₂ is E, X₃ is S, X₄ is S,        and X₅ is Q.        E37. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three complementarity determining regions (CDRs) from a        heavy chain variable region (e.g., H1, H2 or H3), and/or one,        two, or three CDRs from a light chain variable region (e.g., L1,        L2 or L3):    -   a CDR-H1 comprising the amino acid sequence of DYYMN (SEQ ID NO:        8);    -   a CDR-H2 comprising the amino acid sequence of        WIDPDX₁GX₂TIYX₃X₄X₅X₆X₇G (SEQ ID NO: 167), wherein X₁ can be any        one of: an amino acid, an amino acid other than N, a        conservative substitution of N, N, or Q; X₂ can be any one of:        an amino acid, an amino acid other than N, a conservative        substitution of N, N, or Q; X₃ can be any one of: an amino acid,        an amino acid other than D, a conservative substitution of D, D,        or E; X₄ can be any one of: an amino acid, an amino acid other        than P, a conservative substitution of P, P, Q, D, or A; X₅ can        be any one of: an amino acid, an amino acid other than K, a        conservative substitution of K, K, S, or A; X₆ can be any one        of: an amino acid, an amino acid other than F, a conservative        substitution of F, F, or V; and X₇ can be any one of: an amino        acid, an amino acid other than Q, a conservative substitution of        Q, Q, or K;    -   a CDR-H3 comprising the amino acid sequence of RLLMDY (SEQ ID        NO: 10);    -   a CDR-L1 comprising the amino acid sequence of        RSTKSX₈X₉HFNGNX₁₀YLF (SEQ ID NO: 168), wherein X₈ an be any one        of: an amino acid, an amino acid other than L, a conservative        substitution of L, L, or I; X₉ can be any one of: an amino acid,        an amino acid other than L, a conservative substitution of L, L,        or S; and X₁₀ can be any one of: an amino acid, an amino acid        other than T, a conservative substitution of T, T, or S;    -   a CDR-L2 comprising the amino acid sequence of YX₁₁X₁₂SX₁₃LX₁₄S        (SEQ ID NO: 169), wherein X₁₁ can be any one of: an amino acid,        an amino acid other than Y, a conservative substitution of Y, Y,        or A; X₁₂ can be any one of: an amino acid, an amino acid other        than M, a conservative substitution of M, M, or A; X₁₃ can be        any one of: an amino acid, an amino acid other than N, a        conservative substitution of N, N, or S; and X₁₄ can be any one        of: an amino acid, an amino acid other than A, a conservative        substitution of A, A, or Q; and/or    -   a CDR-L3 comprising the amino acid sequence of        X₁₅QSX₁₆X₁₇X₁₈PX₁₉T (SEQ ID NO: 170), wherein X₁₅ can be any one        of: an amino acid, an amino acid other than M, a conservative        substitution of M, M, or Q; X₁₆ can be any one of: an amino        acid, an amino acid other than L, a conservative substitution of        L, L, or Y; X₁₇ can be any one of: an amino acid, an amino acid        other than E, a conservative substitution of E, E, or S; X₁₈ can        be any one of: an amino acid, an amino acid other than Y, a        conservative substitution of Y, Y, or T; and X₁₉ can be any one        of: an amino acid, an amino acid other than F, a conservative        substitution of F, F, L, or W;    -   e.g., wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and        CDR-L3 do not comprise the amino acid sequences of SEQ ID NOs:        22, 23, 24, 25, 26, and 27, respectively, or SEQ ID NOs: 22, 23,        24, 71, 72, and 73, respectively, optionally wherein:    -   X₁ is Q, X₂ is N, X₃ is E, X₄ is P, X₅ is K, X₆ is F, and X₇ is        Q,    -   X₈ is L, X₉ is S, and X₁₀ is T,    -   X₁₁ is Y, X₁₂ is M, X₁₃ is S, and X₁₄ is A, and/or    -   X₁₅ is Q, X₁₆ is L, X₁₇ is E, X₁₈ is Y, and X₁₉ is F.        E38. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three complementarity determining regions (CDRs) from a        heavy chain variable region (e.g., H1, H2 or H3), and/or one,        two, or three CDRs from a light chain variable region (e.g., L1,        L2 or L3):    -   a CDR-H1 comprising the amino acid sequence of DYYMN (SEQ ID NO:        8);    -   a CDR-H2 comprising the amino acid sequence of        WIDPDX₁GNTIYX₂PKX₃QG (SEQ ID NO: 171), wherein X₁ can be any one        of: an amino acid, an amino acid other than N, a conservative        substitution of N, N, or Q; X₂ can be any one of: an amino acid,        an amino acid other than D, a conservative substitution of D, D,        or E; and X₃ can be any one of: an amino acid, an amino acid        other than F, a conservative substitution of F, F, or V;    -   a CDR-H3 comprising the amino acid sequence of RLLMDY (SEQ ID        NO: 10);    -   a CDR-L1 comprising the amino acid sequence of RSTKSLX₄HFNGNTYLF        (SEQ ID NO: 172), wherein X₄ can be any one of: an amino acid,        an amino acid other than L, a conservative substitution of L, L,        or S;    -   a CDR-L2 comprising the amino acid sequence of YYX₅SX₆LAS (SEQ        ID NO: 173), wherein X₅ can be any one of: an amino acid, an        amino acid other than M, a conservative substitution of M, M, or        A; and X₆ can be any one of: an amino acid, an amino acid other        than N, a conservative substitution of N, N, or S; and/or    -   a CDR-L3 comprising the amino acid sequence of X₇QSX₈EYPFT (SEQ        ID NO: 174), wherein X₇ can be any one of: an amino acid, an        amino acid other than M, a conservative substitution of M, M, or        Q; and X₈ can be any one of: an amino acid, an amino acid other        than L, a conservative substitution of L, L, or Y;    -   e.g., wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and        CDR-L3 do not comprise the amino acid sequences of SEQ ID NOs:        22, 23, 24, 25, 26, and 27, respectively, or SEQ ID NOs: 22, 23,        24, 71, 72, and 73, respectively, optionally wherein:    -   X₁ is Q, X₂ is E, and X₃ is F,    -   X₄ is 5,    -   X₅ is M and X₆ is S, and/or    -   X₇ is Q and X₈ is L.        E39. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a VH region comprising        the amino acid sequence of SEQ ID NO: 6, wherein the CDR        sequences are as defined according to Kabat.        E40. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a VL region comprising        the amino acid sequence of SEQ ID NO: 7, wherein the CDR        sequences are as defined according to Kabat.        E41. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a VH region comprising        the amino acid sequence of SEQ ID NO: 6 and one, two, or three        of the CDR-L1 sequences from a VL region comprising the amino        acid sequence of SEQ ID NO: 7, wherein the CDR sequences are as        defined according to Kabat.        E42. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a polypeptide encoded by        the nucleic acid sequence of SEQ ID NO: 189, 190 or 191, wherein        the CDR sequences are as defined according to Kabat.        E43. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a polypeptide encoded by        the nucleic acid sequence of SEQ ID NO: 185 or 186, wherein the        CDR sequences are as defined according to Kabat.        E44. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a polypeptide encoded by        the nucleic acid sequence of SEQ ID NO: 189, 190 or 191 and one,        two, or three of the CDR sequences from a polypeptide encoded by        the nucleic acid sequence of SEQ ID NO: 185 or 186, wherein the        CDR sequences are as defined according to Kabat.        E45. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one        or more complementarity determining regions (CDRs) selected        from:    -   (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:        14,        -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO:            15,        -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO:            16,        -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO:            17,        -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO:            18, and        -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO:            19; or    -   (ii) a CDR-H1 comprising at least one amino acid alteration, but        not more than two, three or four alterations (e.g., a        substitution, deletion, or insertion, e.g., conservative        substitution) relative to SEQ ID NO: 14,        -   a CDR-H2 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 15,        -   a CDR-H3 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 16,        -   a CDR-L1 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 17,        -   a CDR-L2 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 18, or        -   a CDR-L3 comprising at least one amino acid alteration, but            not more than two, three or four alterations (e.g., a            substitution, deletion, or insertion, e.g., conservative            substitution) relative to SEQ ID NO: 19, optionally wherein:        -   any of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3 do            not comprise the amino acid sequence of any of        -   (a) SEQ ID NOs: 28, 29, 30, 31, 32, and 33, respectively, or        -   (b) SEQ ID NOs: 28, 29, 30, 74, 75, and 76, respectively.            E46. An isolated antibody, or antigen-binding fragment            thereof, that specifically binds to human αvβ8 integrin,            comprising one or more complementarity determining regions            (CDRs) selected from:    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 17,        and    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 18.        E47. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E45 or E46, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15,        and    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 16.        E48. The isolated antibody, or antigen-binding fragment thereof,        of any of embodiments E45-E47, comprising:    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 17,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 18,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19.        E49. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 14,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 15,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 16,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 17,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 18,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19.        E50. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of GFNIKDYYMN (SEQ        ID NO: 14);    -   a CDR-H2 comprising the amino acid sequence of WIDPDX₁GN (SEQ ID        NO: 135), wherein X₁ can be any one of: an amino acid, an amino        acid other than N, a conservative substitution of N, N, or Q;    -   a CDR-H3 comprising the amino acid sequence of RLLMDY (SEQ ID        NO: 16);    -   a CDR-L1 comprising the amino acid sequence of STKSLX₂HFNGNTYL        (SEQ ID NO: 136), wherein X₂ can be any one of: an amino acid,        an amino acid other than L, a conservative substitution of L, L,        or S;    -   a CDR-L2 comprising the amino acid sequence of YYMSX₃ (SEQ ID        NO: 137), wherein X₃ can be any one of: an amino acid, an amino        acid other than N, a conservative substitution of N, N, or S;        and    -   a CDR-L3 comprising the amino acid sequence of QSLEYPFT (SEQ ID        NO: 19);    -   e.g., wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and        CDR-L3 do not comprise the amino acid sequences of SEQ ID NOs:        28, 29, 30, 31, 32, and 33, respectively, or SEQ ID NOs: 28, 29,        30, 74, 75, and 76, respectively.        E51. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E50, wherein X₁ is Q, X₂ is S, and X₃ is S.        E52. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of GFNIX₁DYYMN (SEQ        ID NO: 175), wherein X₁ can be any one of: an amino acid, an        amino acid other than K, a conservative substitution of K, K, or        A;    -   a CDR-H2 comprising the amino acid sequence of WIDPDX₂GX₃ (SEQ        ID NO: 176), wherein X₂ can be any one of: an amino acid, an        amino acid other than N, a conservative substitution of N, N, or        Q; and X₃ can be any one of: an amino acid, an amino acid other        than N, a conservative substitution of N, N, or Q;    -   a CDR-H3 comprising the amino acid sequence of RLLMDY (SEQ ID        NO: 16);    -   a CDR-L1 comprising the amino acid sequence of STKSX₄X₅HFNGNX₆YL        (SEQ ID NO: 177), wherein X₄ can be any one of: an amino acid,        an amino acid other than L, a conservative substitution of L, L,        or I; X₅ can be any one of: an amino acid, an amino acid other        than L, a conservative substitution of L, L, or S; and X₆ can be        any one of: an amino acid, an amino acid other than T, a        conservative substitution of T, T, or S;    -   a CDR-L2 comprising the amino acid sequence of YX₇X₈SX₉ (SEQ ID        NO: 178), wherein X₇ can be any one of: an amino acid, an amino        acid other than Y, a conservative substitution of Y, Y, or A; X₈        can be any one of: an amino acid, an amino acid other than M, a        conservative substitution of M, M, or A; and X₉ can be any one        of: an amino acid, an amino acid other than N, a conservative        substitution of N, N, or S; and    -   a CDR-L3 comprising the amino acid sequence of QSX₁₀X₁₁X₁₂PX₁₃T        (SEQ ID NO: 197), wherein X₁₀ can be any one of: an amino acid,        an amino acid other than L, a conservative substitution of L, L,        or Y; X₁₁ can be any one of: an amino acid, an amino acid other        than E, a conservative substitution of E, E, or S; X₁₂ can be        any one of: an amino acid, an amino acid other than Y, a        conservative substitution of Y, Y, or T; and X₁₃ can be any one        of: an amino acid, an amino acid other than F, a conservative        substitution of F, F, L, or W;    -   e.g., wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and        CDR-L3 do not comprise the amino acid sequences of SEQ ID NOs:        28, 29, 30, 31, 32, and 33, respectively, or SEQ ID NOs: 28, 29,        30, 74, 75, and 76, respectively, optionally wherein:    -   X₁ is Q, X₂ is N, X₃ is E, X₄ is P, X₅ is K, X₆ is F, and X₇ is        Q,    -   X₈ is L, X₉ is S, and X₁₀ is T,    -   X₁₁ is Y, X₁₂ is M, X₁₃ is S, and X₁₄ is A, and/or    -   X₁₅ is Q, X₁₆ is L, X₁₇ is E, X₁₈ is Y, and X₁₉ is F.        E53. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of GFNIKDYYMN (SEQ        ID NO: 14);    -   a CDR-H2 comprising the amino acid sequence of WIDPDX₁GN (SEQ ID        NO: 135), wherein X₁ can be any one of: an amino acid, an amino        acid other than N, a conservative substitution of N, N, or Q;    -   a CDR-H3 comprising the amino acid sequence of RLLMDY (SEQ ID        NO: 16);    -   a CDR-L1 comprising the amino acid sequence of STKSLX₂HFNGNTYL        (SEQ ID NO: 136), wherein X₂ can be any one of: an amino acid,        an amino acid other than L, a conservative substitution of L, L,        or S;    -   a CDR-L2 comprising the amino acid sequence of YYX₃SX₄ (SEQ ID        NO: 179), wherein X₃ can be any one of: an amino acid, an amino        acid other than M, a conservative substitution of M, M, or A;        and X₄ can be any one of: an amino acid, an amino acid other        than N, a conservative substitution of N, N, or S; and    -   a CDR-L3 comprising the amino acid sequence of QSX₅EYPFT (SEQ ID        NO: 180), wherein X₅ can be any one of: an amino acid, an amino        acid other than L, a conservative substitution of L, L, or Y;    -   e.g., wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and        CDR-L3 do not comprise the amino acid sequences of SEQ ID NOs:        28, 29, 30, 31, 32, and 33, respectively, or SEQ ID NOs: 28, 29,        30, 74, 75, and 76, respectively, optionally wherein:    -   X₁ is Q,    -   X₂ is S,    -   X₃ is M and X₄ is S, and/or    -   X₅ is L.        E54. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a VH region comprising        the amino acid sequence of SEQ ID NO: 6, wherein the CDR        sequences are as defined according to Chothia.        E55. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a VL region comprising        the amino acid sequence of SEQ ID NO: 7, wherein the CDR        sequences are as defined according to Chothia.        E56. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a VH region comprising        the amino acid sequence of SEQ ID NO: 6 and one, two, or three        of the CDR-L1 sequences from a VL region comprising the amino        acid sequence of SEQ ID NO: 7, wherein the CDR sequences are as        defined according to Chothia.        E57. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a polypeptide encoded by        the nucleic acid sequence of SEQ ID NO: 189, 190 or 191, wherein        the CDR sequences are as defined according to Chothia.        E58. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a polypeptide encoded by        the nucleic acid sequence of SEQ ID NO: 185 or 186, wherein the        CDR sequences are as defined according to Chothia.        E59. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one,        two, or three of the CDR sequences from a polypeptide encoded by        the nucleic acid sequence of SEQ ID NO: 189, 190 or 191 and one,        two, or three of the CDR sequences from a polypeptide encoded by        the nucleic acid sequence of SEQ ID NO: 185, 186, wherein the        CDR sequences are as defined according to Chothia.        E60. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        comprises a VH framework region (e.g., one, two, three, or four        of FR1, FR2, FR3, or FR4) comprising an amino acid sequence        having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or        preferably 100% sequence identity to a VH framework region of a        VH region comprising the amino acid sequence of any one of SEQ        ID NOs: 6, 34-46, 88-91, or 93.        E61. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        comprises a VL framework region (e.g., one, two, three, or four        of FR1, FR2, FR3, or FR4) comprising an amino acid sequence        having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or        preferably 100% sequence identity to a VL framework region of a        VL region comprising the amino acid sequence of any one of SEQ        ID NOs: 7, 47-69, or 92.        E62. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        comprises a VH framework region (e.g., one, two, three, or four        of FR1, FR2, FR3, or FR4) comprising an amino acid sequence        having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or        100% sequence identity to the germline amino acid sequence of        IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69, or IGHV3-48.        E63. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        comprises a VL framework region (e.g., one, two, three, or four        of FR1, FR2, FR3, or FR4) comprising an amino acid sequence        having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or        100% sequence identity to the germline amino acid sequence of        IGKV1-39, IGKV2-28, IGKV2-30, IGKV4-1, or IGKV3-11.        E64. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        comprises a VH framework region (e.g., one, two, three, or four        of FR1, FR2, FR3, or FR4) comprising an amino acid sequence        comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, or 23 substitutions relative to a VH        framework region of a VH region comprising the amino acid        sequence of any one of SEQ ID NOs: 6, 34-46, 88-91, or 93.        E65. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        comprises a VL framework region (e.g., one, two, three, or four        of FR1, FR2, FR3, or FR4) comprising an amino acid sequence        comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, or 23 substitutions relative to a VL        framework region of a VL region comprising the amino acid        sequence of any one of SEQ ID NOs: 7, 47-69, or 92.        E66. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        comprises a VH framework region (e.g., one, two, three, or four        of FR1, FR2, FR3, or FR4) comprising an amino acid sequence        comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, or 23 substitutions relative to the        germline amino acid sequence of IGHV3-07, IGHV1-46, IGHV3-23,        IGHV3-30, IGHV1-69, or IGHV3-48.        E67. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        comprises a VL framework region (e.g., one, two, three, or four        of FR1, FR2, FR3, or FR4) comprising an amino acid sequence        comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, or 23 substitutions relative to the        germline amino acid sequence of IGKV1-39, IGKV2-28, IGKV2-30,        IGKV4-1, or IGKV3-11.        E68. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, comprising a murine IgG1 Fc        region comprising a substitution at one or more positions        selected from E233, E318, K320, and R322 (e.g., E233P, E318A,        K320A, and R322A), e.g., wherein the murine IgG1 Fc region        comprises one or more of the E233P, E318A, K320A, and R322A        substitutions, as numbered according to the Eu numbering scheme        (see e.g., U.S. Pat. No. 5,624,821).        E69. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, comprising a human IgG1 Fc        region comprising a substitution at one or more positions        selected from L234, L235, and G237 (e.g., L234A, L235A, and        G237A), e.g., wherein the human IgG1 Fc region comprises one or        more of the L234A, L235A, and G237A substitutions, as numbered        according to the Eu numbering scheme.        E70. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        further comprises a VH region comprising a variant of the        germline VH amino acid sequence of IGHV3-07, IGHV1-46, IGHV3-23,        IGHV3-30, IGHV1-69, or IGHV3-48, wherein the VH region comprises        one or more substitutions at positions T28, F29, A49, R72, N74,        A75, and/or L79 (e.g., one or more substitutions selected from        T28N, F29I, A49G, R72A, N74T, A75S and L79A), as numbered        according to the amino acid sequence of SEQ ID NO: 127,        optionally wherein the VH region comprises the substitutions:    -   (i) T28N and F29I;    -   (ii) T28N, F29I, and R72A;    -   (iii) T28N, F29I, R72A, A49G, and L79A;    -   (iv) T28N, F29I, R72A, N74T, and A75S; or    -   (v) T28N, F29I, R72A, A49G, L79A, N74T, and A75S,    -   wherein (i)-(v) are as numbered according to the amino acid        sequence of SEQ ID NO: 127, optionally wherein:    -   the VH region comprises the substitutions T28N, F29I, R72A,        A49G, L79A, N74T, and A75S, numbered according to the amino acid        sequence of SEQ ID NO: 127.        E71. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        further comprises a VH region comprising one or more (e.g., 2,        3, 4, 5, 6, or all) of the following:    -   (a) an Asn at position 28,    -   (b) an Ile at position 29,    -   (c) a Gly at position 49,    -   (d) an Ala at position 72,    -   (e) a Thr at position 74,    -   (f) a Ser at position 75, and    -   (g) an Ala at position 79, numbered according to the amino acid        sequence of SEQ ID NO: 127, optionally wherein the VH region        comprises:    -   (i) an Asn at position 28 and an Ile at position 29;    -   (ii) an Asn at position 28, an Ile at position 29, and an Ala at        position 72;    -   (iii) an Asn at position 28, an Ile at position 29, an Ala at        position 72, a Gly at position 49, and an Ala at position 79;    -   (iv) an Asn at position 28, an Ile at position 29, an Ala at        position 72, a Thr at position 74, and a Ser at position 75; or    -   (v) an Asn at position 28, an Ile at position 29, an Ala at        position 721, a Gly at position 49, an Ala at position 79, a Thr        at position 74, and a Ser at position 75, numbered according to        the amino acid sequence of SEQ ID NO: 127, optionally wherein:    -   the VH region comprises an Asn at position 28, an Ile at        position 29, an Ala at position 72, a Gly at position 49, an Ala        at position 79, a Thr at position 743, and a Ser at position 75,        numbered according to the amino acid sequence of SEQ ID NO: 127.        E72. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        further comprises a VL region comprising a variant of the        germline VL amino acid sequence of IGKV1-39, IGKV2-28, IGKV2-30,        IGKV4-1, or IGKV3-11, wherein the VH region comprises one or        more substitutions at positions Y36 and/or L46 (e.g., Y36F        and/or L46R), as numbered according to the amino acid sequence        of SEQ ID NO: 128, optionally wherein the VL region comprises        the substitutions:    -   (i) L46R; or    -   (ii) L46R and Y36F,    -   wherein (i)-(v) are as numbered according to the amino acid        sequence of SEQ ID NO: 128, optionally wherein the VL region        comprises the substitution L46R, numbered according to the amino        acid sequence of SEQ ID NO: 128.        E73. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, wherein the antibody        further comprises a VL region comprising one or both of the        following:    -   (a) a Tyr at position 36, and    -   (b) a Leu at position 46, numbered according to the amino acid        sequence of SEQ ID NO:128, optionally wherein the VL region        comprises:    -   (i) a Leu at position 46; or    -   (ii) a Leu at position 46 and a Tyr at position 36, numbered        according to the amino acid sequence of SEQ ID NO:128,        optionally wherein:    -   the VL region comprises a Leu at position 46, numbered according        to the amino acid sequence of SEQ ID NO:128.        E74. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising one        or more CDRs of any of the preceding embodiments, wherein the        one or more CDRs comprise at least one amino acid alteration,        but not more than two, three or four alterations (e.g., a        substitution, deletion, or insertion, e.g., conservative        substitution); wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1,        CDR-L2, and CDR-L3 do not comprise the amino acid sequences of        SEQ ID NOs: 22, 23, 24, 25, 26, and 27, respectively, SEQ ID        NOs: 22, 23, 24, 71, 72, and 73, respectively, SEQ ID NOs: 28,        29, 30, 31, 32, and 33, respectively, or SEQ ID NOs: 28, 29, 30,        74, 75, and 76, respectively.        E75. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, further comprising a VH        region comprising an amino acid sequence set forth in Table 1.        E76. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, further comprising a VL        region comprising an amino acid sequence set forth in Table 1.        E77. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, comprising a VH region        comprising an amino acid sequence having at least 80%, 85%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence        identity to any one of SEQ ID NOs: 6, 34-46, 88-91, or 93.        E78. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E77, comprising a VH region comprising the amino        acid sequence of SEQ ID NO: 6.        E79. The isolated antibody, or antigen-binding fragment thereof,        of any of the preceding embodiments, comprising a VL region        comprising an amino acid sequence having at least 80%, 85%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence        identity to any one of SEQ ID NOs: 7, 47-69, or 92.        E80. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E79, comprising a VL region having at least 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%        sequence identity (e.g., 100%) to the amino acid sequence of SEQ        ID NO: 7.        E81. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a VH        region comprising an amino acid sequence having at least 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%        sequence identity to any one of SEQ ID NOs: 6, 34-46, 88-91, or        93.        E82. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E81, comprising a VH region having at least 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%        sequence identity (e.g., 100%) to the amino acid sequence of SEQ        ID NO: 6.        E83. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E81 or E82, further comprising a VL region having        at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        99%, or 100% sequence identity (e.g., 100%) to the amino acid        sequence of SEQ ID NO: 7.        E84. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E81, comprising a VH region comprising the amino        acid sequence of SEQ ID NO: 6.        E85. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E84, comprising a VL region comprising the amino        acid sequence of SEQ ID NO: 7.        E86. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a VL        region comprising an amino acid sequence having at least 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%        sequence identity to any one of SEQ ID NOs: 7, 47-69, or 92.        E87. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E86, comprising a VL region having at least 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%        sequence identity (e.g., 100%) to the amino acid sequence of SEQ        ID NO: 7.        E88. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E86 or E87, further comprising a VH region having        at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        99%, or 100% sequence identity (e.g., 100%) to the amino acid        sequence of SEQ ID NO: 6.        E89. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E86, comprising a VL region comprising an amino        acid sequence selected from SEQ ID NOs: 7, 47-69, or 92.        E90. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a VH        region comprising the amino acid sequence of SEQ ID NO: 39,        wherein one or more amino acid residues of said SEQ ID NO: 39        comprise one or more amino acid substitutions selected from        K30A, N55Q, N57Q, D61E, P62A, K63A, and F64V, numbered according        to SEQ ID NO: 39.        E91. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a VH        region comprising at least one of the following:    -   (a) an Ala at position 30    -   (b) a Gln at position 55,    -   (c) a Gln at position 57,    -   (d) a Glu at position 61,    -   (e) an Ala at position 62,    -   (f) an Ala at position 63, and    -   (g) a Val at position 64, numbered according to SEQ ID NO: 39.        E92. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E90, wherein said SEQ ID NO: 39 comprises:    -   (i) N55Q and D61E; or    -   (ii) N55Q, D61E, and F64V, numbered according to SEQ ID NO: 39,        optionally wherein said SEQ ID NO: 39 comprises N55Q and D61E        substitutions, numbered according to SEQ ID NO: 39.        E93. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E91, wherein the VH region comprises:    -   (i) a Gln at position 55 and a Glu at position 61; or    -   (ii) a Gln at position 55, a Glu at position 61, and a Val at        position 64, numbered according to SEQ ID NO: 39, optionally        wherein the VH region comprises a Gln at position 55 and a Glu        at position 61, numbered according to SEQ ID NO: 39.        E94. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E90 or E92, further comprising a VL region        comprising the amino acid sequence of SEQ ID NO: 47, wherein one        or more amino acid residues of said SEQ ID NO: 47 comprise one        or more amino acid substitutions selected from L305, Y55A, M56A,        N58S, A60Q, M94Q, L97Y, F101L, F101W, and Q105G, or any        combination thereof, numbered according to SEQ ID NO: 47,        optionally wherein one or more amino acid residues of said SEQ        ID NO: 47 comprise one or more amino acid substitutions selected        from L305, M56A, N58S, M94Q, L97Y, and Q105G.        E95. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E91 or E93, further comprising a VL region        comprising at least one of the following:    -   (a) a Ser at position 30,    -   (b) an Ala at position 55,    -   (c) an Ala at position 56,    -   (d) a Ser at position 58,    -   (e) a Gln at position 60,    -   (f) a Gln at position 94,    -   (g) a Tyr at position 97,    -   (h) a Leu at position 101,    -   (i) a Trp at position 101, and    -   (j) a Gly at position 105, numbered according to SEQ ID NO: 47,        optionally wherein the VL region comprises at least one of the        following:    -   (a) a Ser at position 30,    -   (b) an Ala at position 56,    -   (c) a Ser at position 58,    -   (d) a Gln at position 94,    -   (e) a Tyr at position 97, and    -   (f) a Gly at position 105.        E96. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E94, wherein said SEQ ID NO: 47 comprises a L305,        M56A, N58S, M94Q, L97Y, and/or Q105G substitution, numbered        according to SEQ ID NO: 47.        E97. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E95, wherein the VL region comprises a Ser at        position 30, an Ala at position 56, a Ser at position 58, a Gln        at position 94, a Tyr at position 97, and/or a Gly at position        105, numbered according to SEQ ID NO: 47.        E98. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E94, wherein said SEQ ID NO: 47 comprises a L305,        N58S, M94Q, and/or Q105G substitution, numbered according to SEQ        ID NO: 47, optionally wherein said SEQ ID NO: 47 comprises all        of L305, N58S, M94Q, and Q105G substitutions.        E99. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E95, wherein the VL region comprises a Ser at        position 30, a Ser at position 58, a Gln at position 94, and/or        a Gly at position 105, numbered according to SEQ ID NO: 47,        optionally wherein the VL region comprises all of: a Ser at        position 30, a Ser at position 58, a Gln at position 94, and a        Gly at position 105.        E100. The isolated antibody, or antigen-binding fragment        thereof, of embodiment E94, wherein said SEQ ID NO: 39 comprises        N55Q and D61E substitutions, numbered according to SEQ ID NO:        39, and said SEQ ID NO: 47 comprises L305, N58S, M94Q, and Q105G        substitutions, numbered according to SEQ ID NO: 47.        E101. The isolated antibody, or antigen-binding fragment        thereof, of embodiment E95, wherein the VH region comprises a        Gln at position 55 and a Glu at position 61, numbered according        to SEQ ID NO: 39, and the VL region comprises a Ser at position        30, a Ser at position 58, a Gln at position 94, and a Gly at        position 105, numbered according to SEQ ID NO: 47.        E102. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a VL        region comprising the amino acid sequence of SEQ ID NO: 47,        wherein one or more amino acid residues of said SEQ ID NO: 47        comprise one or more amino acid substitutions selected from        L305, Y55A, M56A, N58S, A60Q, M94Q, L97Y, F101L, F101W, and        Q105G, or any combination thereof (e.g., all of L30S, M56A,        N58S, M94Q, L97Y, and Q105G), numbered according to SEQ ID NO:        47, optionally wherein one or more amino acid residues of said        SEQ ID NO: 47 comprise one or more amino acid substitutions        selected from L305, M56A, N58S, M94Q, L97Y, and Q105G.        103. The isolated antibody, or antigen-binding fragment thereof,        of embodiment E102, wherein said SEQ ID NO: 47 comprises a L305,        N58S, M94Q, and/or Q105G substitution, numbered according to SEQ        ID NO: 47.        E104. The isolated antibody, or antigen-binding fragment        thereof, of embodiment E102, further comprising a VH region        comprising the amino acid sequence of SEQ ID NO: 39, wherein the        sequence of SEQ ID NO: 39 comprises one or more amino acid        substitutions selected from K30A, N55Q, N57Q, D61E, P62A, K63A,        and F64V, or any combination thereof, numbered according to SEQ        ID NO: 39.        E105. The isolated antibody, or antigen-binding fragment        thereof, of embodiment E104, wherein said SEQ ID NO: 39        comprises N55Q and D61E substitutions.        E106. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, comprising a heavy        chain comprising an amino acid sequence having at least 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%        sequence identity to SEQ ID NO: 2 or 3.        E107. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, further comprising        a light chain comprising an amino acid sequence having at least        80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or        100% sequence identity to SEQ ID NO: 5.        E108. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a        heavy chain variable region comprising the amino acid sequence        of SEQ ID NO: 6 and a light chain variable region comprising the        amino acid sequence of SEQ ID NO: 7.        E109. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a        heavy chain comprising the amino acid sequence of SEQ ID NO: 2        or 3, and a light chain comprising the amino acid sequence of        SEQ ID NO: 5.        E110. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising        (a) a light chain comprising the amino acid sequence of SEQ ID        NO: 5 and        (b) a heavy chain comprising the amino acid sequence of SEQ ID        NO: 3, with or without a C-terminal lysine residue.        E111. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a        heavy chain comprising an amino acid sequence encoded by the        insert of the plasmid deposited with the ATCC and having the        Accession Number PTA-124917, a light chain comprising an amino        acid sequence encoded by the insert of the plasmid deposited        with the ATCC and having the Accession Number PTA-124918, or        both.        E112. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a        heavy chain comprising an amino acid sequence having at least        80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or        100% (e.g., 100%) sequence identity to SEQ ID NO: 2 or 3,        optionally wherein the heavy chain comprises the amino acid        sequence of SEQ ID NO: 2.        E113. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a        light chain comprising an amino acid sequence having at least        80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or        100% (e.g., 100%) sequence identity to SEQ ID NO: 5, optionally        wherein the light chain comprises the amino acid sequence of SEQ        ID NO: 5, optionally wherein the isolated antibody, or        antigen-binding fragment thereof, further comprises a heavy        chain comprising the amino acid sequence of SEQ ID NO: 2.        E114. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a        heavy chain comprising an amino acid sequence comprising SEQ ID        NO: 2, and a light chain comprising an amino acid sequence        comprising SEQ ID NO: 5.        E115. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising a        heavy chain comprising an amino acid sequence comprising SEQ ID        NO: 3, and a light chain comprising an amino acid sequence        comprising SEQ ID NO: 5.        E116. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;        and    -   wherein the antibody further comprises a VH framework region        (e.g., one, two, three or all four of FR1, FR2, FR3, or FR4)        comprising an amino acid sequence having at least 80%, 85%, 87%        90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% sequence        identity to a VH framework region of a VH region comprising the        amino acid sequence of any one of SEQ ID NOs: 6, 34-46, 88-91,        or 93, or an amino acid sequence having at least one, but less        than twenty alterations, e.g., an amino acid substitution or        deletion, of the amino acid sequence of the entire VH framework        region (including FR1, FR2, FR3 and FR4).        E117. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;        and    -   wherein the antibody further comprises a VL framework region        (e.g., FR1, FR2, FR3, or FR4) comprising an amino acid sequence        having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or        preferably 100% sequence identity to a VL framework region of a        VL region comprising the amino acid sequence of any one of SEQ        ID NOs: 7, 47-69, or 92, or an amino acid sequence having at        least one, two, three, four, five, six, seven, ten, fifteen, but        less than twenty alterations, e.g., an amino acid substitution        or deletion, of the amino acid sequence of the entire VL        framework region (including FR1, FR2, FR3, and FR4).        E118. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;        and        wherein the antibody comprises a VH framework region (e.g., FR1,        FR2, FR3, or FR4) comprising an amino acid sequence having at        least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or 100%        sequence identity to a VH framework region within the germline        amino acid sequence of IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30,        IGHV1-69, or IGHV3-48, or an amino acid sequence having at least        one, two, three, four, five, six, seven, ten, fifteen, but less        than twenty alterations, e.g., an amino acid substitution or        deletion, of the amino acid sequence of the entire VH framework        region (including FR1, FR2, FR3, and FR4).        E119. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;        and        wherein the antibody comprises a VH region comprising a variant        of the germline VH amino acid sequence of IGHV3-07, IGHV1-46,        IGHV3-23, IGHV3-30, IGHV1-69, or IGHV3-48, wherein the VH region        comprises one or more substitutions at positions T28, F29, A49,        R71, N73, A74, and/or L78 (e.g., one or more substitutions        selected from T28N, F29I, A49G, R72A, N74T, A75S and L79A), as        numbered according to the amino acid sequence of SEQ ID NO: 127,        optionally wherein the VH region comprises the substitutions:    -   (i) T28N and F29I;    -   (ii) T28N, F29I, and R72A;    -   (iii) T28N, F29I, R72A, A49G, and L79A;    -   (iv) T28N, F29I, R72A, N74T, and A75S; or    -   (v) T28N, F29I, R72A, A49G, L79A, N74T, and A75S,    -   wherein (i)-(v) are as numbered according to the amino acid        sequence of SEQ ID NO: 127, optionally wherein:    -   the VH region comprises the substitutions T28N, F29I, R72A,        A49G, L79A, N74T, and A75S, numbered according to the amino acid        sequence of SEQ ID NO: 127.        E120. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;        and        wherein the antibody comprises a VH region comprising one or        more (e.g., 2, 3, 4, 5, 6, or all) of the following:    -   (a) an Asn at position 28,    -   (b) an Ile at position 29,    -   (c) a Gly at position 49,    -   (d) an Ala at position 72,    -   (e) a Thr at position 74,    -   (f) a Ser at position 75, and    -   (g) an Ala at position 79, numbered according to the amino acid        sequence of SEQ ID NO: 127, optionally wherein the VH region        comprises:    -   (i) an Asn at position 28 and an Ile at position 29;    -   (ii) an Asn at position 28, an Ile at position 29, and an Ala at        position 72;    -   (iii) an Asn at position 28, an Ile at position 29, an Ala at        position 72, a Gly at position 49, and an Ala at position 79;    -   (iv) an Asn at position 28, an Ile at position 29, an Ala at        position 72, a Thr at position 74, and a Ser at position 75; or    -   (v) an Asn at position 28, an Ile at position 29, an Ala at        position 72, a Gly at position 49, an Ala at position 79, a Thr        at position 74, and a Ser at position 75, numbered according to        the amino acid sequence of SEQ ID NO: 127, optionally wherein:    -   the VH region comprises an Asn at position 28, an Ile at        position 29, an Ala at position 72, a Gly at position 49, an Ala        at position 79, a Thr at position 74, and a Ser at position 75,        numbered according to amino acid sequence of SEQ ID NO: 127.        E121. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;        and        wherein the antibody comprises a VL framework region (e.g., FR1,        FR2, FR3, or FR4) comprising an amino acid sequence having at        least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or 100%        sequence identity to the germline amino acid sequence of        IGKV1-39, IGKV2-28, IGKV2-30, IGKV4-1, or IGKV3-11, or an amino        acid sequence having at least one, two, three, four, five, six,        seven, ten, fifteen, but less than twenty alternations, e.g., an        amino acid substitution or deletion, of the amino acid sequence        of the entire VL framework region (including FR1, FR2, FR3, and        FR4).        E122. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;        and        wherein the antibody comprises a VL region comprising a variant        of the germline VL amino acid sequence of IGKV1-39, IGKV2-28,        IGKV2-30, IGKV4-1, or IGKV3-11, wherein the VL region comprises        one or more substitutions at positions Y36 and/or L46 (e.g.,        Y36F and/or L46R), as numbered according to the amino acid        sequence of SEQ ID NO: 128, optionally wherein the VL region        comprises the substitutions:    -   (i) L46R; or    -   (ii) L46R and Y36F,    -   wherein (i) and (ii) are numbered according to the amino acid        sequence of SEQ ID NO: 128, optionally wherein the VL region        comprises the substitution L46R, numbered according to the amino        acid sequence of SEQ ID NO: 128.        E123. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;        and        wherein the antibody comprises a VL region comprising one or        both of the following:    -   (a) a Tyr at position 36, and    -   (b) a Leu at position 46, numbered according to the amino acid        sequence of SEQ ID NO: 128, optionally wherein the VL region        comprises:    -   (i) a Leu at position 46; or    -   (ii) a Leu at position 46 and a Tyr at position 36, numbered        according to the amino acid sequence of SEQ ID NO: 128,        optionally wherein:    -   the VL region comprises a Leu at position 46, numbered according        to the amino acid sequence of SEQ ID NO: 128.        E124. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;        and        further comprising a murine IgG1 Fc region comprising one or        more substitutions selected from positions E233, E318, K320, and        R322 (e.g., E233P, E318A, K320A, and R322A) as numbered        according to the Eu numbering scheme, e.g., relative to murine        IgG1 Fc set forth in Table 1.        E125. The isolated antibody, or antigen-binding fragment        thereof, of embodiment E124, wherein the murine IgG1 Fc region        comprises the E233P, E318A, K320A, and R322A substitutions as        numbered according to the Eu numbering scheme, e.g., relative to        murine IgG1 Fc set forth in Table 1.        E126. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27;        and        further comprising a human IgG1 Fc region comprising one or more        substitutions selected from positions L234, L235, and G237        (e.g., L234A, L235A, and G237A) as numbered according to the Eu        numbering scheme, e.g., relative to human IgG1 Fc set forth in        Table 1.        E127. The isolated antibody, or antigen-binding fragment        thereof, of embodiment E126, wherein the human IgG1 Fc region        comprises the L234A, L235A, and G237A substitutions, as numbered        according to the Eu numbering scheme, e.g., relative to human        IgG1 Fc set forth in Table 1.        E128. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73;        and    -   wherein the antibody further comprises a VH framework region        (e.g., one, two, three, or four or FR1, FR2, FR3, or FR4)        comprising an amino acid sequence having at least 80%, 85%, 87%        90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% sequence        identity to a VH framework region of a VH region comprising the        amino acid sequence of any one of SEQ ID NOs: 6, 34-46, 88-91,        or 93.        E129. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73;        and    -   wherein the antibody further comprises a VL framework region        (e.g., one, two, three, or four or FR1, FR2, FR3, or FR4)        comprising an amino acid sequence having at least 80%, 85%, 87%        90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% sequence        identity to a VL framework region of a VL region comprising the        amino acid sequence of any one of SEQ ID NOs: 7, 47-69, or 92.        E130. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73;        and        wherein the antibody further comprises a VH framework region        (e.g., one, two, three, or four or FR1, FR2, FR3, or FR4)        comprising an amino acid sequence having at least 80%, 85%, 87%        90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to a VH        framework region within the germline amino acid sequence of        IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69, or IGHV3-48.        E131. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73;        and        wherein the antibody further comprises a VL framework region        (e.g., one, two, three, or four or FR1, FR2, FR3, or FR4)        comprising an amino acid sequence having at least 80%, 85%, 87%        90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to the        germline amino acid sequence of IGKV1-39, IGKV2-28, IGKV2-30,        IGKV4-1, or IGKV3-11.        E132. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73;        and        further comprising a murine IgG1 Fc region comprising one or        more substitutions selected from positions E233, E318, K320, and        R322 (e.g., E233P, E318A, K320A, and R322A) as numbered        according to the Eu numbering scheme, e.g., relative to murine        IgG1 Fc set forth in Table 1.        E133. The isolated antibody, or antigen-binding fragment        thereof, of embodiment E132, wherein the murine IgG1 Fc region        comprises E233P, E318A, K320A, and R322A substitutions as        numbered according to the Eu numbering scheme, e.g., relative to        murine IgG1 Fc set forth in Table 1.        E134. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 71,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 72,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 73;        and        further comprising a human IgG1 Fc region comprising L234A,        L235A, and G237A substitutions as numbered according to the Eu        numbering scheme, e.g., relative to human IgG1 Fc set forth in        Table 1.        E135. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33;        and    -   wherein the antibody further comprises a VH framework region        (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4)        comprising an amino acid sequence having at least 80%, 85%, 87%        90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% sequence        identity to a VH framework region of a VH region comprising the        amino acid sequence of any one of SEQ ID NOs: 6, 34-46, 88-91,        or 93.        E136. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 74,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 75,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 76;        and    -   wherein the antibody further comprises a VH framework region        (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4)        comprising an amino acid sequence having at least 80%, 85%, 87%        90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% sequence        identity to a VH framework region of a VH region comprising the        amino acid sequence of any one of SEQ ID NOs: 6, 34-46, 88-91,        or 93.        E137. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33;        and    -   wherein the antibody further comprises a VL framework region        (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4)        comprising an amino acid sequence having at least 80%, 85%, 87%        90%, 92%, 93%, 95%, 97%, 98%, or preferably 100% sequence        identity to a VL framework region of a VL region comprising the        amino acid sequence of any one of SEQ ID NOs: 7, 47-69, or 92.        E138. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33;        and        wherein the antibody further comprises a VH framework region        (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4)        comprising an amino acid sequence having at least 80%, 85%, 87%        90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to the        germline amino acid sequence of IGHV3-07, IGHV1-46, IGHV3-23,        IGHV3-30, IGHV1-69, or IGHV3-48.        E139. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to human αvβ8 integrin, comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33;        and        wherein the antibody further comprises a VL framework region        (e.g., one, two, three, or four of FR1, FR2, FR3, or FR4)        comprising an amino acid sequence having at least 80%, 85%, 87%        90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to the        germline amino acid sequence of IGKV1-39, IGKV2-28, IGKV2-30,        IGKV4-1, or IGKV3-11.        E140. An isolated antibody, or antigen-binding fragment thereof,        comprising:    -   a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28,    -   a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29,    -   a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30,    -   a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31,    -   a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32,        and    -   a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33;        and        further comprising a human IgG1 Fc region comprising one or more        substitutions selected from positions L234, L235, and G237        (e.g., L234A, L235A, and G237A) as numbered according to the Eu        numbering scheme, e.g., relative to human IgG1 Fc set forth in        Table 1.        E141. The isolated antibody, or antigen-binding fragment        thereof, of embodiment E140, wherein the human IgG1 Fc region        comprises L234A, L235A, and G237A substitutions as numbered        according to the Eu numbering scheme, e.g., relative to human        IgG1 Fc set forth in Table 1.        E142. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, wherein the        antibody is a multispecific antibody (e.g., a bispecific        antibody).        E143. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, wherein the        antibody is a multivalent antibody (e.g., a bivalent antibody).        E144. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, wherein the        antibody is a humanized antibody, a human antibody, a murine        antibody, chimeric antibody, or a camelid antibody.        E145. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds to αvβ8 integrin, comprising a VH region        and a VL region, wherein the VH region and VL region comprise        the amino acid sequences of:    -   (i) SEQ ID NOs: 6 and 7, respectively, or an amino acid sequence        having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or        preferably 100% sequence identity thereto;    -   (ii) SEQ ID NOs: 34 and 65, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (iii) SEQ ID NOs: 34 and 62, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (iv) SEQ ID NOs: 34 and 66, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (v) SEQ ID NOs: 34 and 63, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (vi) SEQ ID NOs: 34 and 64, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (vii) SEQ ID NOs: 37 and 65, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (viii) SEQ ID NOs: 37 and 62, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (ix) SEQ ID NOs: 37 and 66, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (x) SEQ ID NOs: 37 and 63, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xi) SEQ ID NOs: 37 and 64, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xii) SEQ ID NOs: 36 and 65, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xiii) SEQ ID NOs: 36 and 62, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xiv) SEQ ID NOs: 36 and 66, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xv) SEQ ID NOs: 36 and 63, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xvi) SEQ ID NOs: 36 and 64, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xvii) SEQ ID NOs: 35 and 65, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xviii) SEQ ID NOs: 35 and 62, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xix) SEQ ID NOs: 35 and 66, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xx) SEQ ID NOs: 35 and 63, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxi) SEQ ID NOs: 35 and 64, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxii) SEQ ID NOs: 38 and 65, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxiii) SEQ ID NOs: 38 and 62, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxiv) SEQ ID NOs: 38 and 66, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxv) SEQ ID NOs: 38 and 63, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxvi) SEQ ID NOs: 38 and 64, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxvii) SEQ ID NOs: 20 and 21, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxviii) SEQ ID NOs: 88 and 47, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxix) SEQ ID NOs: 89 and 47, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxx) SEQ ID NOs: 90 and 47, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxxi) SEQ ID NOs: 90 and 92, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxxii) SEQ ID NOs: 39 and 47, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxxiii) SEQ ID NOs: 6 and 67, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxxiv) SEQ ID NOs: 6 and 68, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxxv) SEQ ID NOs: 6 and 69, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxxvi) SEQ ID NOs: 93 and 67, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxxvii) SEQ ID NOs: 93 and 68, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto;    -   (xxxviii) SEQ ID NOs: 93 and 69, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto; or    -   (xxxix) SEQ ID NOs: 93 and 7, respectively, or an amino acid        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or preferably 100% sequence identity thereto, optimally        wherein:    -   the VH region and VL region comprise the amino acid sequences of    -   (i) SEQ ID NOs: 6 and 7, respectively;    -   (ii) SEQ ID NOs: 34 and 65, respectively;    -   (iii) SEQ ID NOs: 34 and 62, respectively;    -   (iv) SEQ ID NOs: 34 and 66, respectively;    -   (v) SEQ ID NOs: 34 and 63, respectively;    -   (vi) SEQ ID NOs: 34 and 64, respectively;    -   (vii) SEQ ID NOs: 37 and 65, respectively;    -   (viii) SEQ ID NOs: 37 and 62, respectively;    -   (ix) SEQ ID NOs: 37 and 66, respectively;    -   (x) SEQ ID NOs: 37 and 63, respectively;    -   (xi) SEQ ID NOs: 37 and 64, respectively;    -   (xii) SEQ ID NOs: 36 and 65, respectively;    -   (xiii) SEQ ID NOs: 36 and 62, respectively;    -   (xiv) SEQ ID NOs: 36 and 66, respectively;    -   (xv) SEQ ID NOs: 36 and 63, respectively;    -   (xvi) SEQ ID NOs: 36 and 64, respectively;    -   (xvii) SEQ ID NOs: 35 and 65, respectively;    -   (xviii) SEQ ID NOs: 35 and 62, respectively;    -   (xix) SEQ ID NOs: 35 and 66, respectively;    -   (xx) SEQ ID NOs: 35 and 63, respectively;    -   (xxi) SEQ ID NOs: 35 and 64, respectively;    -   (xxii) SEQ ID NOs: 38 and 65, respectively;    -   (xxiii) SEQ ID NOs: 38 and 62, respectively;    -   (xxiv) SEQ ID NOs: 38 and 66, respectively;    -   (xxv) SEQ ID NOs: 38 and 63, respectively;    -   (xxvi) SEQ ID NOs: 38 and 64, respectively;    -   (xxvii) SEQ ID NOs: 20 and 21, respectively;    -   (xxviii) SEQ ID NOs: 88 and 47, respectively;    -   (xxix) SEQ ID NOs: 89 and 47, respectively;    -   (xxx) SEQ ID NOs: 90 and 47, respectively;    -   (xxxi) SEQ ID NOs: 90 and 92, respectively;    -   (xxxii) SEQ ID NOs: 39 and 47, respectively;    -   (xxxiii) SEQ ID NOs: 6 and 67, respectively;    -   (xxxiv) SEQ ID NOs: 6 and 68, respectively;    -   (xxxv) SEQ ID NOs: 6 and 69, respectively;    -   (xxxvi) SEQ ID NOs: 93 and 67, respectively;    -   (xxxvii) SEQ ID NOs: 93 and 68, respectively;    -   (xxxviii) SEQ ID NOs: 93 and 69, respectively; or    -   (xxxix) SEQ ID NOs: 93 and 7, respectively.        E146. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which comprises or        has a heavy chain constant region (Fc) chosen from, e.g., the        heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM,        IgA1, IgA2, IgD, and IgE.        E147. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, wherein the        antibody belongs to an isotype chosen from IgG1, IgG2, IgG3,        IgG4, or any variant thereof.        E148. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, wherein the        antibody comprises a heavy chain constant region of IgG1 or IgG2        (e.g., human IgG1 or human IgG2).        E149. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which comprises or        has a heavy chain constant region is human IgG1.        E150. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which comprises or        has a light chain constant region chosen from, e.g., the light        chain constant regions of kappa or lambda.        E151. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which comprises or        has a kappa (e.g., human kappa) light chain constant region.        E152. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which comprises an        Fc region of the heavy chain having an altered hinge region to        reduce effector cell function.        E153. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which has reduced        antibody dependent cellular cytotoxicity (ADCC) and/or reduced        complement dependent cytotoxicity (CDC).        E154. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which comprises a        hinge region having a substitution at at least one position of        L234, L235 or G237, e.g., as compared to a human IgG1, numbered        according to the Eu numbering scheme.        E155. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, comprising a human        IgG1 Fc region comprising at least one substitution selected        from L234A, L235A, and G237A, numbered according to the Eu        numbering scheme.        E156. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which has a hinge        region comprising the amino acid sequence of        EPKSCDKTHTCPPCPAPEAAGAP (SEQ ID NO: 126).        E157. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which is altered        to remove an immunogenic T-cell epitope.        E158. The isolated antibody, or antigen-binding fragment        thereof, of embodiment E157, which comprises a VL comprising at        least one substitution selected from the group consisting of        L305, N58S, M56A, M94Q, L97Y and Q105G, numbered according to        SEQ ID NO: 47.        E159. The antibody, or antigen-binding fragment thereof, of any        of the preceding embodiments, wherein said antibody, or        antigen-binding fragment thereof, has at least one of the        following properties:    -   (i) a binding affinity, expressed as KD, for human αvβ8 integrin        that is less than the murine antibody ADWA11, e.g., less than        536 pM;    -   (ii) a KD for human αvβ8 integrin that is less than or equal to        100 pM for purified human αvβ8 integrin;    -   (iii) a KD for mouse αvβ8 integrin that is less than 100 pM;    -   (iv) a KD for cynomolgus monkey αvβ8 integrin that is less 100        pM;    -   (v) a KD for rat αvβ8 integrin that is about 160 pM;    -   (vi) approximately equivalent affinity for at least two, three,        or all of human, cynomolgus, mouse, and rat αvβ8 integrin, e.g.,        with a KD that is less than 100 pM, e.g., as determined using a        Biacore affinity assay;    -   (vii) an IC50 for inhibiting TGFβ transactivation that is less        than 183 pM;    -   (viii) an IC50 for inhibiting TGFβ transactivation in U251 cells        that is about 100 pM to about 300 pM;    -   (ix) an EC50 for U251 cells of about 100 pM to about 400 pM pM;    -   (x) an EC50 for C8-S cells of about 110 pM to about 180 pM;    -   (xi) at least one predicted human pharmacokinetic (PK) parameter        chosen from:        -   a. a clearance from central compartment (CL) of about            0.12-0.15 mL/h/kg;        -   b. an inter-compartmental distribution clearance (CLF) of            about 0.15-0.51 mL/h/kg;        -   c. a volume of distribution for the central compartment (V1)            of about 36-39 mL/kg;        -   d. a volume of distribution for the peripheral compartment            (V2) of about 21-33 mL/kg; and/or        -   e. a terminal half-life (t_(1/2)) of about 12-17 days; and    -   (xii) no detectable binding to human Fcγ receptors or C1q.        E160. The antibody, or antigen-binding fragment thereof, of any        of the preceding embodiments, wherein said antibody, or        antigen-binding fragment thereof, has at least one of the        following properties:    -   (i) binds specifically to αvβ8 integrin but not to other        integrins;    -   (ii) reduces an interaction between αvβ8 integrin and Latency        Associated Peptide (LAP);    -   (iii) reduces TGF-β signaling;    -   (iv) effectively blocks the αvβ8 integrin-mediated TGFβ        activation with an IC50≤10 nM;    -   (v) has a comparable Kd (within 5-fold) towards a non-human        primate (NHP) orthologue;    -   (vi) selectivity binds human αvβ8 and does not detectably bind a        homologue of αvβ8 (e.g., αvβ1, αvβ3, αvβ5 and αvβ6);    -   (vii) causes growth suppression and/or complete tumor regression        in a human subject or an animal model of cancer, for example,        squamous cell carcinoma, breast cancer, and/or colon cancer,        alone or in combination with an immunomodulatory agent, e.g., a        modulators of checkpoint inhibitors, e.g., inhibitors of PD-1,        PD-L1, CTLA-4, or an agonist of a stimulatory molecule, e.g.,        4-1BB;    -   (viii) causes growth suppression and/or complete tumor        regression in an animal model for a cancer in combination with        an anti-cancer therapy, e.g., radiotherapy;    -   (ix) shows at least 60% reduction in tumor growth in a syngeneic        tumor graft model, e.g., when administered at ≤10 mg/kg alone or        in combination with an immunomodulatory agent (e.g., an        inhibitor of PD-1, PD-L1, CTLA-4);    -   (x) increases an anti-tumor response in the presence of one or        more immunomodulators, e.g., an antagonist of a checkpoint        inhibitor or an agonist of a checkpoint activator, e.g., an        antagonist of PD-1, PD-L1, or CTLA-4, or an activator of an        immune response, e.g., 4-1BB agonist, when administered to a        subject, e.g., a mouse or human subject;    -   (xi) has an efficacy that is not dependent upon the expression        of αvβ8 integrin in a tumor model;    -   (xii) can increase the abundance of CD8+ GzmB+ T cells in the        tumor microenvironment, e.g., as a monotherapy;    -   (xiii) shows a decrease, e.g., at least a >80% decrease, in        tumor growth when used in combination with an antagonist of a        checkpoint inhibitor (e.g., an anti-PD-1 or anti-PD-L1        antibody), e.g., in a syngeneic model of squamous cell        carcinoma, breast cancer, and/or colon cancer;    -   (xiv) shows a statistically significant improvement in overall        survival of a subject, as determined by a Kaplan-Meier analysis;    -   (xv) shows a high degree of thermal stability;    -   (xvi) shows minimal aggregation at high concentration; and    -   (xvii) may show reproducible expression and purity in        large-scale manufacturing conditions.        E161. The antibody, or antigen-binding fragment thereof, of any        of the preceding embodiments, wherein said antibody, or        antigen-binding fragment thereof, has at least one of the        following properties:    -   (i) a binding affinity, expressed as KD, for human αvβ8 integrin        that is less than the KD for the murine antibody ADWA11, e.g.,        less than 536 pM;    -   (ii) a KD for human αvβ8 integrin that is less than or equal to        100 pM for purified human αvβ8 integrin;    -   (iii) a KD for mouse αvβ8 integrin that is less than 100 pM;    -   (iv) a KD for cynomolgus monkey αvβ8 integrin that is less than        100 pM;    -   (v) a KD for rat αvβ8 integrin that is about 160 pM;    -   (vi) approximately equivalent affinity for at least two, three,        or all of human, cynomolgus, mouse, and rat αvβ8 integrin, e.g.,        with a KD that is less than 100 pM, as determined using a        Biacore affinity assay;    -   (vii) an IC50 for inhibiting TGFβ transactivation that is less        than 183 pM;    -   (viii) an IC50 for inhibiting TGFβ transactivation in U251 cells        of about 100 pM to about 300 pM;    -   (ix) an EC50 for U251 cells of about 126 pM with a standard        deviation of plus or minus 34 pM;    -   (x) an EC50 for U251 cells of about 256 pM with a standard        deviation of plus or minus 115 pM;    -   (xi) an EC50 for U251 cells of about 80 pM to about 400 pM;    -   (xii) an EC50 for C8-S cells of about 115 pM;    -   (xiii) an EC50 for C8-S cells of about 145 pM with a standard        deviation of plus or minus 23.7 pM;    -   (xiv) an EC50 for C8-S cells of about 110 pM to about 180 pM;    -   (xv) at least one predicted human pharmacokinetic (PK) parameter        chosen from:        -   a. a clearance from central compartment (CL) of about            0.12-0.15 mL/h/kg;        -   b. an inter-compartmental distribution clearance (CLF) of            about 0.15-0.51 mL/h/kg;        -   c. a volume of distribution for the central compartment (V1)            of about 36-39 mL/kg;        -   d. a volume of distribution for the peripheral compartment            (V2) of about 21-33 1 mL/kg; and/or        -   e. a terminal half-life (t_(1/2)) of about 12-17 days; and    -   (xvi) no detectable binding to human Fcγ receptors or C1q.        E162. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which binds human        αvβ8 integrin with a K_(D) less than or equal to 100 pM for        purified human αvβ8 integrin.        E163. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which binds human        αvβ8 integrin with a K_(D) less than 536 pM.        E164. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which inhibits        TGFβ activation with an IC50 less than 183 pM.        E165. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which inhibits        TGFβ activation with an IC50 of 100 pM to about 300 pM.        E166. The isolated antibody, or antigen-binding fragment        thereof, of any of the preceding embodiments, which inhibits        TGFβ activation in U251 cells with an IC50 of 199+/−93.6 pM.        E167. A pharmaceutical composition comprising the antibody, or        antigen-binding fragment thereof, of any of the preceding        embodiments, and a pharmaceutically acceptable carrier or        excipient.        E168. A nucleic acid molecule that encodes the antibody, or        antigen-binding fragment thereof, of any of embodiments E1-E166.        E169. A nucleic acid molecule comprising:    -   (i) a nucleotide sequence having at least 80%, 85%, 87% 90%,        92%, 93%, 95%, 97%, 98%, or 100% sequence identity to SEQ ID NO:        1, 183, 189 or 191 and encoding a heavy chain;    -   (ii) a nucleotide sequence having at least 80%, 85%, 87% 90%,        92%, 93%, 95%, 97%, 98%, or 100% sequence identity to SEQ ID NO:        190 and encoding a heavy chain variable region;    -   (iii) a nucleotide sequence having at least 80%, 85%, 87% 90%,        92%, 93%, 95%, 97%, 98%, or 100% sequence identity to SEQ ID NO:        192 or 193 and encoding a heavy chain constant region; or    -   (iv) a nucleotide sequence having at least 80%, 85%, 87% 90%,        92%, 93%, 95%, 97%, 98%, or 100% sequence identity to a nucleic        acid sequence of the insert of the plasmid deposited with the        ATCC and having Accession Number PTA-124917.        E170. A nucleic acid molecule comprising:    -   (i) a nucleotide sequence having at least 80%, 85%, 87% 90%,        92%, 93%, 95%, 97%, 98%, or 100% sequence identity to SEQ ID NO:        4 or 185 and encoding a light chain;    -   (ii) a nucleotide sequence having at least 80%, 85%, 87% 90%,        92%, 93%, 95%, 97%, 98%, or 100% sequence identity to SEQ ID        NO:186 and encoding a light chain variable region;    -   (iii) a nucleotide sequence having at least 80%, 85%, 87% 90%,        92%, 93%, 95%, 97%, 98%, or 100% sequence identity to SEQ ID NO:        194 and encoding a light chain constant region; or    -   (iv) a nucleotide sequence having at least 80%, 85%, 87% 90%,        92%, 93%, 95%, 97%, 98%, or 100% sequence identity to a nucleic        acid sequence of the insert of the plasmid deposited with the        ATCC and having Accession Number PTA-124918.        E171. A nucleic acid molecule comprising a nucleotide sequence        having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or        100% sequence identity to SEQ ID NO: 1 or 183 and a nucleotide        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or 100% sequence identity to SEQ ID NO: 4.        E172. A nucleic acid molecule comprising a nucleotide sequence        having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or        100% sequence identity to SEQ ID NO: 189 or 191 and a nucleotide        sequence having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%,        98%, or 100% sequence identity to SEQ ID NO: 185.        E173. A vector comprising the nucleic acid molecule of any of        embodiments E168-E172.        E174. A host cell comprising the nucleic acid molecule of any of        embodiments E168-E172 or the vector of embodiment E173.        E175. The host cell of embodiment E174, wherein the host cell is        a mammalian cell, e.g., a human cell.        E176. The host cell of embodiment E175, wherein the host cell is        a CHO cell, a COS cell, a HEK-293 cell, an NS0 cell, a PER.C6®        cell, or an Sp2.0 cell.        E177. A method of making an isolated antibody, or        antigen-binding fragment thereof, that specifically binds to        human αvβ8 integrin, comprising culturing the host cell of any        one of embodiments E174-E176, under conditions wherein said        antibody or antigen-binding fragment is expressed by said host        cell.        E178. The method of embodiment E177, further comprising        isolating the antibody or antigen-binding fragment thereof.        E179. A method of reducing TGFβ signaling in a subject in need        thereof, the method comprising administering to a subject in        need thereof a therapeutically effective amount of the antibody,        or antigen-binding fragment thereof, of any of embodiments        E1-E166, or the pharmaceutical composition of any one of        embodiment E167.        E180. A method of reducing αvβ8 integrin activity in a subject        in need thereof, the method comprising administering to a        subject in need thereof a therapeutically effective amount of        the antibody, or antigen-binding fragment thereof, of any of        embodiments E1-E166, or the pharmaceutical composition of        embodiment E167.        E181. A method of treating a disease, disorder, or condition        associated with or mediated by aberrant (e.g., increased) TGFβ        signaling, comprising administering to a subject in need thereof        a therapeutically effective amount of the antibody, or        antigen-binding fragment thereof, of any of embodiments E1-E166,        or the pharmaceutical composition of embodiment E167.        E182. A method of inducing an anti-tumor response in a subject,        comprising administering to a subject in need thereof a        therapeutically effective amount of the antibody, or        antigen-binding fragment thereof, of any of embodiments E1-E166,        or the pharmaceutical composition of any one of embodiment E167,        optionally, wherein the antibody, or antigen-binding fragment        thereof, is administered in combination with a second therapy,        optionally wherein the antibody, or antigen-binding fragment        thereof, and the second therapy are administered simultaneously,        sequentially, or separately, optionally wherein:    -   (i) the antibody, or antigen-binding fragment thereof, is        administered prior to the administration of the second therapy,        or    -   (ii) the antibody, or antigen-binding fragment thereof, is        administered after the administration of the second therapy.        E183. The antibody, or antigen-binding fragment thereof, of any        of embodiments E1-E166, or the pharmaceutical composition of        embodiment E167, for use in reducing the activity of αvβ8        integrin in a subject.        E184. A method of treating a cancer, comprising administering to        a subject in need thereof a therapeutically effective amount of        the antibody, or antigen-binding fragment thereof, of any of        embodiments E1-E166, or the pharmaceutical composition of        embodiment E167.        E185. The method of embodiment E184, wherein said antibody, or        antigen-binding fragment thereof, has at least one of the        following properties:    -   (i) binds specifically to αvβ8 integrin (e.g., αvβ8 integrin        from human, mouse, cynomolgus monkey, and/or rat);    -   (ii) reduce an interaction between αvβ8 integrin and Latency        Associated Peptide (LAP);    -   (iii) reduces TGF-β signaling;    -   (iv) blocks the αvβ8 integrin-mediated TGFβ activation with an        IC50≤10 nM;    -   (v) has a comparable Kd (within 5-fold) towards a non-human        primate (NHP) orthologue;    -   (vi) selectivity binds human αvβ8 and does not detectably bind a        homologue of αvβ8 (e.g., αvβ1, αvβ3, αvβ5 and αvβ6);    -   (vii) causes growth suppression and/or complete tumor regression        in an animal model for a cancer chosen from, for example,        squamous cell carcinoma, breast, and colon cancer, alone or in        combination with an immunomodulatory agent, e.g., a modulators        of checkpoint inhibitors, e.g., inhibitors of PD-1, CTLA-4, or        an agonist of a stimulatory molecule, e.g., 4-1BB;    -   (viii) causes growth suppression and/or complete tumor        regression in an animal model for a cancer in combination with        an anti-cancer therapy, e.g., radiotherapy;    -   (ix) shows at least 60% reduction in tumor growth in a syngeneic        tumor graft model, e.g., when administered at ≤10 mg/kg;    -   (x) increases an anti-tumor response in the presence of one or        more immunomodulators, e.g., an antagonist of a checkpoint        inhibitor, e.g., an antagonist of PD-1 or CTLA-4, or an        activator of an immune response, e.g., 4-1BB agonist, when        administered to a subject, e.g., a mouse or human subject;    -   (xi) has an efficacy that is not dependent upon the expression        of αvβ8 integrin in a tumor model;    -   (xii) is sufficient to increase the abundance of CD8+ GzmB+ T        cell in the tumor microenvironment, e.g., as a monotherapy;    -   (xiii) shows at least an 80% decrease, in tumor growth when used        in combination with an antagonist of a checkpoint inhibitor        (e.g., an anti-PD-1 or anti-PD-L1 antibody), e.g., in a        syngeneic model of squamous cell carcinoma, breast cancer,        and/or colon cancer;    -   (xiv) shows a statistically significant improvement in overall        survival of a subject, e.g., a human or a mouse, as determined        by a Kaplan-Meier analysis;    -   (xv) shows a high degree of thermal stability;    -   (xvi) shows minimal aggregation at high concentration; and    -   (xvii) may show reproducible expression and purity in        large-scale manufacturing conditions.        E186. The method of embodiment E184 or E185, wherein said        antibody, or antigen-binding fragment thereof, has at least one        of the following properties:    -   (i) a binding affinity, expressed as KD, for human αvβ8 integrin        that is less than the KD for the murine antibody ADWA11, e.g.,        less than 536 pM;    -   (ii) a KD for human αvβ8 integrin that is less than or equal to        100 pM for purified human αvβ8 integrin;    -   (iii) a KD for mouse αvβ8 integrin that is less than 100 pM;    -   (iv) a KD for cynomolgus monkey αvβ8 integrin that is less than        100 pM;    -   (v) a KD for rat αvβ8 integrin that is about 160 pM;    -   (vi) shows approximately equivalent affinity for at least two,        three, or all of human, cynomolgus, mouse, and rat αvβ8        integrin, e.g., with a KD that is less than 100 pM, e.g., as        determined using a Biacore affinity assay.    -   (vii) an IC50 for inhibiting TGFβ transactivation that is less        than 183 pM;    -   (viii) an IC50 for inhibiting TGFβ transactivation in U251 cells        of about 199+/−93.6 pM;    -   (ix) an IC50 for inhibiting TGFβ transactivation that is about        100 pM to about 300 pM.    -   (x) an EC50 for U251 cells of about 126 pM with a standard        deviation of plus or minus 34 pM;    -   (xi) an EC50 for U251 cells of about 256 pM with a standard        deviation of plus or minus 115 pM;    -   (xii) an EC50 for U251 cells of about 80 pM to about 400 pM;    -   (xiii) an EC50 for C8-S cells of about 115 pM;    -   (xiv) an EC50 for C8-S cells of about 145 pM with a standard        deviation of plus or minus 23.7 pM;    -   (xv) an EC50 for C8-S cells of about 110 pM to about 180 pM;    -   (xvi) at least one predicted human pharmacokinetic (PK)        parameter chosen from:        -   a. a clearance from central compartment (CL) of about            0.12-0.15 mL/h/kg;        -   b. an inter-compartmental distribution clearance (CLF) of            about 0.15-0.51 mL/h/kg;        -   c. a volume of distribution for the central compartment (V1)            of about 36-39 mL/kg;        -   d. a volume of distribution for the peripheral compartment            (V2) of about 21-33 mL/kg; and/or        -   e. a terminal half-life (t_(1*2)) of about 12-17 days; and    -   (xvii) shows no detectable binding to human Fcγ receptors or        C1q.        E187. The method of any of embodiments E184-E186, wherein said        antibody, or antigen-binding fragment thereof, is according to        any of embodiments E1-E166, or the pharmaceutical composition of        embodiment E167.        E188. The method of any of embodiments E184-E187, wherein the        antibody, or antigen-binding fragment thereof, is administered        in an amount sufficient to increases CD45+ cell, CD3+ T cell,        CD4+ T cell, CD8+ T cells, and/or Granzyme B expressing cell        infiltration.        E189. The method of any of embodiments E184-E188, wherein the        antibody, or antigen-binding fragment thereof, is administered        in an amount sufficient to increases CD8+ T cells infiltration.        E190. The method of any of embodiments E184-E189, wherein the        antibody, or antigen-binding fragment thereof, is administered        in an amount sufficient to increase the expression of Granzyme B        on CD8+ T cells.        E191. The method of any of embodiments E184-E190, wherein the        antibody, or antigen-binding fragment thereof, is administered        in an amount sufficient to increase the accumulation of        inflammatory macrophages having an elevated level of Ly6G        expression.        E192. The method of any of embodiments E184-E191, wherein the        antibody, or antigen-binding fragment thereof, is administered        in an amount sufficient to increase the accumulation of        CD45+CD11b+CD11c-Ly6G-Ly6C^(high)CD206^(low) inflammatory        macrophages.        E193. The method of any of embodiments E184-E192, wherein the        antibody, or antigen-binding fragment thereof, is administered        in an amount sufficient to increase the response a second        therapy.        E194. The method of any of embodiments E184-E193, wherein the        efficacy of the antibody, or antigen-binding fragment thereof,        when administered to an animal tumor model is not dependent upon        expression of αvβ8 integrin by the tumor model.        E195. The method of any of embodiments E184-E194, wherein the        administration of said antibody, or antigen-binding fragment        thereof, occurs in combination with a second therapy.        E196. The method of embodiment E195, wherein the second therapy        comprises an anti-cancer therapy, a cytotoxic or cytostatic        agent, e.g., a chemotherapeutic agent, a hormone treatment, a        vaccine, and/or an immunotherapy.        E197. The method of embodiment E195 or E196, wherein the second        therapy is or comprises surgery, radiation, cryosurgery, and/or        thermotherapy.        E198. The method of any of embodiments E195-E197, wherein the        second therapy comprises a modulator, e.g., an inhibitor or an        agonist, of an immune checkpoint molecule, optionally wherein        the second therapy is or comprises a modulator of an immune        checkpoint molecule selected from the group consisting of PD1,        PD-L1, 4-1BB, OX40, CTLA-4, PD-L2, TIM-3, LAG-3, VISTA, CD160,        BTLA, TIGIT, 2B4, TGFβ, LAIR1 and a combination thereof.        E199. The method of embodiment E198, wherein the inhibitor of an        immune checkpoint molecule is an inhibitor of PD1, PD-L1,        CTLA-4, PD-L2, TIM-3, LAG-3, VISTA, BTLA, TIGIT, 2B4, TGFβ, or        LAIR1.        E200. The method of embodiment E199, wherein the inhibitor of an        immune checkpoint molecule is an inhibitor of PD1, e.g., an        antibody against PD1.        E201. The method of embodiment E199, wherein the inhibitor of an        immune checkpoint molecule is an inhibitor CTLA-4, e.g., an        antibody against CTLA-4 or a soluble CTLA-4 fusion.        E202. The method of embodiment E195-E201, wherein the second        therapy comprises an agonist of a costimulatory molecule.        E203. The method of embodiment E202, wherein the agonist of a        costimulatory molecule is selected from at least one of 4-1BB        (CD137), OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS        (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C,        SLAMF7, NKp80, CD160, or B7-H3.        E204. The method of embodiment E202, wherein the agonist of a        costimulatory molecule is a 41-BB agonist.        E205. The method of embodiment E195-E204, wherein the second        therapy comprises an inhibitor of PARP1 (e.g., olaparib,        rucaparib, niraparib, veliparib, iniparib, talazoparib,        3-aminobenzamide, CEP 9722, E7016, BSI-201, KU-0059436,        AG014699, MK-4827, or BGB-290).        E206. The method of embodiment E184-E205, wherein the cancer is        selected from the group consisting of a solid tumor, a        hematological cancer (e.g., leukemia, lymphoma, myeloma, e.g.,        multiple myeloma), and a metastatic lesion.        E207. The method of embodiment E206, wherein the cancer is a        solid tumor.        E208. The method of embodiment E206 or E207, wherein the cancer        is a solid tumor and is chosen from a malignancy, e.g., sarcomas        and carcinomas, e.g., adenocarcinomas of the various organ        systems, such as those affecting the lung (e.g., a non-small        cell lung cancer (NSCLC)), breast, ovarian, lymphoid,        gastrointestinal (e.g., colon), anal, genitals and genitourinary        tract (e.g., renal, urothelial, bladder cells, prostate),        pharynx, CNS (e.g., brain, neural or glial cells), head and neck        (e.g., head and neck squamous cell carcinoma (HNSCC), skin        (e.g., melanoma, e.g., an advanced melanoma), pancreas, colon,        rectal, a renal (e.g., a renal cell carcinoma), liver, cancer of        the small intestine and cancer of the esophagus,        gastro-esophageal cancer, thyroid cancer, and cervical cancer.        E209. The method of any of embodiment E184-E208, wherein the        cancer is a lymphoproliferative disease (e.g., a post-transplant        lymphoproliferative disease) or a hematological cancer, T-cell        lymphoma, B-cell lymphoma, a non-Hodgkin lymphoma, or a leukemia        (e.g., a myeloid leukemia or a lymphoid leukemia).        E210. The method of any of embodiment E184-E209, wherein the        cancer is an early, intermediate, late stage or metastatic        cancer.        E211. The method of any of embodiment E184-E210, wherein the        cancer is selected from the group consisting of a renal cell        carcinoma, an ovarian cancer, and a head and neck squamous cell        carcinoma.        E212. The method of embodiment E211, further comprising        administering an inhibitor of a checkpoint inhibitor, e.g., an        inhibitor of PD-1, PD-L1, or CTLA-4.        E213. The method of embodiment E212, wherein the inhibitor of        PD-L1 in not avelumab.        E214. The method of embodiment E211 or E212, wherein the cancer        is a renal cancer, e.g., a renal cell carcinoma (RCC).        E215. The method of embodiment E214, wherein the cancer is a        renal cancer selected from the group consisting of a metastatic        RCC, a clear cell renal cell carcinoma (ccRCC)), a        non-clear-cell renal cell carcinoma (ncRCC), and high risk renal        cell carcinoma.        E216. The method of embodiment E215, wherein the antibody, or        antigen-binding fragment thereof is administered as a 1^(st)        line or 2^(nd) line therapy.        E217. The method of any of embodiments E184-E216, wherein the        antibody, or antigen-binding fragment thereof, is administered        as a 1^(st) line therapy.        E218. The method of any of embodiments E184-E216, wherein the        antibody, or antigen-binding fragment thereof, is administered        as a 2nd line therapy.        E219. The method of any of embodiments E184-E212, wherein the        cancer is an ovarian cancer.        E220. The method of embodiment E219, wherein the second therapy        is an inhibitor of PARP1 (e.g., olaparib, rucaparib, niraparib,        veliparib, iniparib, talazoparib, 3-aminobenzamide, CEP 9722,        E7016, BSI-201, KU-0059436, AG014699, MK-4827, or BGB-290).        E221. The method of embodiment E219, wherein the antibody, or        antigen-binding fragment thereof is administered as a 2^(nd)        line therapy, optionally wherein the subject is        platinum-resistant.        E222. The method of embodiment E219, wherein the antibody, or        antigen-binding fragment thereof is administered as a 1^(st)        line therapy.        E223. The method of any of embodiments E184-E222, wherein the        cancer is a head and neck squamous cell carcinoma.        E224. The method of embodiment E223, wherein the method further        comprises administration of radiation therapy.        E225. The method of embodiment E223 or E224, wherein the cancer        is platinum-resistant and/or recurrent cancer.        E226. The method of any of embodiments E179-E225, wherein said        subject is a human.        E227. The method of any of embodiments E179-E226, comprising        administering said antibody or antigen-binding fragment thereof,        or pharmaceutical composition, intravenously.        E228. The method of any of embodiments E179-E227, comprising        administering said antibody or antigen-binding fragment thereof,        or pharmaceutical composition, subcutaneously.        E229. The method of any of embodiments E179-E228, wherein said        antibody or antigen-binding fragment thereof, or pharmaceutical        composition, is administered about twice a week, once a week,        once every two weeks, once every three weeks, once every four        weeks, once every five weeks, once every six weeks, once every        seven weeks, once every eight weeks, once every nine weeks, once        every ten weeks, twice a month, once a month, once every two        months, once every three months, once every four months, once        every five months, once every six months, once every seven        months, once every eight months, once every nine months, once        every ten months, once every eleven months or once every twelve        months.        E230. The method of any of embodiments E179-E229, wherein the        antibody, or the antigen-binding fragments thereof, is        administered every two weeks, e.g., up to 12 times (e.g., up to        10, 8, 6, 5, 4, or 3 times).        E231. The method of embodiment E230, wherein each administration        comprises 5-10 mg/kg (e.g., 5, 6, 7, 8, 9, or 10 mg/kg) of the        antibody, or the antigen-binding fragments thereof.        E232. The method of embodiment E231, wherein each administration        comprises about 7 mg/kg.        E233. The method of any of embodiments E179-E229, wherein the        antibody, or the antigen-binding fragments thereof, is        administered every four weeks, e.g., up to 6 times (e.g., up to        6, 5, 4, 3, 2, or 1 time).        E234. The method of embodiment E233, wherein each administration        comprises 10-15 mg/kg (e.g., 10, 11, 12, 13, 14, or 15 mg/kg) of        the antibody, or the antigen-binding fragments thereof.        E235. The method of embodiment E234, wherein each administration        comprises about 12 mg/kg.        E236. A method of detecting αvβ8 integrin (e.g., human αvβ8        integrin) in a sample, tissue, or cell using the antibody, or        antigen-binding fragment thereof, of any of embodiments E1-E166,        or the pharmaceutical composition of embodiment E167, comprising        contacting the sample, tissue or cell with the antibody and        detecting the antibody.        E237. A kit comprising the antibody, or antigen-binding fragment        thereof, of any of embodiments E1-E166, or the pharmaceutical        composition of embodiment E167.        E238. The antibody, or antigen-binding fragment thereof, of any        of embodiments E1-E166, or the pharmaceutical composition of        embodiment E167, for use as a medicament, e.g., in any of the        method embodiments described herein.        E239. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds αvβ8 integrin, wherein the antibody or        fragment is at least one antibody or fragment selected from the        group consisting of:    -   (a) an antibody or antigen-binding fragment thereof, comprising        a light chain complementarity determining region 1 (CDR-L1)        comprising the amino acid sequence of SEQ ID NO:11; a CDR-L2        comprising the amino acid sequence of SEQ ID NO:12; a CDR-L3        comprising the amino acid sequence of SEQ ID NO:13; a heavy        chain CDR1 (CDR-H1) comprising the amino acid sequence of SEQ ID        NO:8; a CDR-H2 comprising the amino acid sequence of SEQ ID        NO:9; and a CDR-H3 comprising the amino acid sequence of SEQ ID        NO:10;    -   (b) an antibody or antigen-binding fragment thereof, comprising        a CDR-L1 comprising the amino acid sequence of SEQ ID NO:17; a        CDR-L2 comprising the amino acid sequence of SEQ ID NO:18; a        CDR-L3 comprising the amino acid sequence of SEQ ID NO:19; a        CDR-H1 comprising the amino acid sequence of SEQ ID NO:14; a        CDR-H2 comprising the amino acid sequence of SEQ ID NO:15; and a        CDR-H3 comprising the amino acid sequence of SEQ ID NO:16;    -   (c) an antibody or antigen-binding fragment thereof, comprising        a variable light (VL) region comprising an amino acid sequence        encoded by the insert of the plasmid deposited with the ATCC and        having Accession Number PTA-124918, and a variable heavy (VH)        region comprising an amino acid sequence encoded by the insert        of the plasmid deposited with the ATCC having Accession Number        PTA-124917;    -   (d) an antibody or antigen-binding fragment thereof, comprising        a VL region comprising the amino acid sequence of SEQ ID NO:7,        and a VH region comprising the amino acid sequence of SEQ ID        NO:6;    -   (e) an antibody or antigen-binding fragment thereof, comprising        a VL region comprising the amino acid sequence selected from the        group consisting of SEQ ID NO:62-66, and a VH region comprising        the amino acid sequence selected from the group consisting of        SEQ ID NO:34-38;    -   (f) an antibody or antigen-binding fragment thereof, comprising        a VL region comprising the amino acid sequence selected from the        group consisting of SEQ ID NO:47 and 92, and a VH region        comprising the amino acid sequence selected from the group        consisting of SEQ ID NO:39 and 88-91;    -   (g) an antibody or antigen-binding fragment thereof, comprising        a VL region comprising the amino acid sequence selected from the        group consisting of SEQ ID NO:7 and 67-69, and a VH region        comprising the amino acid sequence selected from the group        consisting of SEQ ID NO:6 and 93;    -   (h) an antibody or antigen-binding fragment thereof, comprising        a VL region comprising the amino acid sequence selected from the        group consisting of SEQ ID NO:7, 47-69 and 92, and a VH region        comprising the amino acid sequence selected from the group        consisting of SEQ ID NO:6, 34-46, 88-91 and 93;    -   (i) an antibody or antigen-binding fragment thereof, comprising        a light chain (LC) region comprising the amino acid sequence of        SEQ ID NO:5, and a heavy chain (HC) region comprising the amino        acid sequence of SEQ ID NO:2;    -   (j) an antibody or antigen-binding fragment thereof, comprising        a LC region comprising the amino acid sequence of SEQ ID NO:5,        and a HC region comprising the amino acid sequence of SEQ ID        NO:3;    -   (k) an antibody or antigen-binding fragment thereof, comprising        a LC region comprising the amino acid sequence of SEQ ID NO:123,        and a HC region comprising the amino acid sequence of SEQ ID        NO:124 or 182;    -   (l) an antibody or antigen-binding fragment thereof, comprising        a VL region encoded by the nucleic acid sequence of SEQ ID        NO:186, and a VH region encoded by the nucleic acid sequence of        SEQ ID NO:190; and    -   (m) an antibody or antigen-binding fragment thereof, comprising        a LC region encoded by the nucleic acid sequence of SEQ ID        NO:185, and a HC region encoded by the nucleic acid sequence of        SEQ ID NO:189 or 191.        E240. The isolated antibody or antigen-binding fragment thereof        of embodiment E239, comprising a VL region comprising the amino        acid sequence of SEQ ID NO:7, and a VH region comprising the        amino acid sequence of SEQ ID NO:6.        E241. The isolated antibody or antigen-binding fragment thereof        of embodiments E239 or E240, comprising a VL region comprising        an amino acid sequence at least 95% identical to SEQ ID NO:7,        and a VH region comprising an amino acid sequence at least 95%        identical to SEQ ID NO:6.        E242. The isolated antibody or antigen-binding fragment thereof        of embodiment E239, comprising a LC region comprising the amino        acid sequence of SEQ ID NO:5, and a HC region comprising the        amino acid sequence of SEQ ID NO:2 or 3.        E243. The isolated antibody or antigen-binding fragment thereof        of embodiments E239 or E242, comprising a LC region comprising        an amino acid sequence at least 95% identical to SEQ ID NO:5,        and a HC region comprising an amino acid sequence at least 95%        identical to SEQ ID NO:2 or 3.        E244. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds αvβ8 integrin, wherein the antibody or        fragment comprises a VH region comprising an amino acid sequence        having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        99%, or 100% sequence identity to an amino acid sequence        selected from the group consisting of SEQ ID NO:6, 34-46, 88-91,        and 93, and/or a VL region comprising an amino acid sequence        having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,        99%, or 100% sequence identity to an amino acid sequence        selected from the group consisting of SEQ ID NOs: 7, 47-69, and        92.        E245. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds αvβ8 integrin, wherein the antibody or        fragment comprises:    -   (i) an antibody HC comprising an amino acid sequence having at        least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%        sequence identity to SEQ ID NO: 2 or 3; and/or    -   (ii) an antibody LC comprising an amino acid sequence having at        least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%        sequence identity to SEQ ID NO: 5.        E246. An isolated antibody that specifically binds αvβ8        integrin, comprising a LC consisting of the amino acid sequence        of SEQ ID NO:5, and HC consisting of the amino acid sequence of        SEQ ID NO:2 or 3.        E247. An isolated antibody that specifically binds αvβ8        integrin, comprising:    -   an antibody VL region comprising the CDR-L1, CDR-L2 and CDR-L3        from the VL region comprising the amino acid sequence of SEQ ID        NO:7; and    -   an antibody VH region comprising the CDR-H1, CDR-H2, and CDR-H3        from the VH region comprising the amino acid sequence of SED ID        NO:6.        E248. The isolated antibody of embodiment E247, comprising an        antibody heavy chain constant region comprising the amino acid        sequence of SEQ ID NO: 181 or 184 and an antibody light chain        constant region comprising the amino acid sequence of SEQ ID NO:        83.        E249. An isolated antibody that specifically binds αvβ8        integrin, comprising:    -   a) an antibody VL region comprising the first, second and third        CDRs from the VL region comprising the amino acid sequence of        SEQ ID NO:7;    -   an antibody VH region comprising the first, second and third        CDRs from the VH region comprising the amino acid sequence of        SEQ ID NO:6;    -   an antibody light chain constant (CL) region comprising the        amino acid sequence of SEQ ID NO:83; and    -   an antibody heavy chain (CH) constant region comprising the        amino acid sequence of SEQ ID NO:181 or 184;    -   b) an antibody VL region comprising an amino acid sequence at        least 95% identical to SEQ ID NO: 7; and an antibody VH region        comprising an amino acid sequence at least 95% identical to SEQ        ID NO:6; or    -   c) an antibody LC region comprising an amino acid sequence at        least 95% identical to SEQ ID NO: 5, and an antibody HC        comprising an amino acid sequence at least 95% identical to SEQ        ID NO: 2 or 3.        E250. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds αvβ8 integrin, comprising an antibody VH        comprising an amino acid sequence encoded by the insert        deposited with the ATCC and having the Accession Number        PTA-124917, and an antibody VL comprising an amino acid sequence        encoded by the insert deposited with the ATCC and having the        Accession Number PTA-124918.        E251. An isolated antibody, or antigen-binding fragment thereof,        that specifically binds αvβ8 integrin, wherein the antibody or        fragment has at least one of the following properties:    -   a. a binding affinity, expressed as KD, for human αvβ8 integrin        that is less than the KD for the murine antibody ADWA11, e.g.,        less than about 536 pM;    -   b. a KD for human αvβ8 integrin that is less than or equal to        about 100 pM;    -   c. a KD for mouse αvβ8 integrin that is less than the KD for the        murine antibody ADWA11, e.g., less than about 489 pM;    -   d. a KD for mouse αvβ8 integrin that is less than about 100 pM;    -   e. a KD for cynomolgus monkey αvβ8 integrin that is less than        the KD for the murine antibody ADWA11, e.g., less than about 507        pM;    -   f. a KD for cynomolgus monkey αvβ8 integrin that is less than or        equal to about 100 pM;    -   g. a KD for rat αvβ8 integrin that is about 160 pM;    -   h. approximately equivalent affinity for at least two, three, or        all of human, cynomolgus, mouse, and rat αvβ8 integrin, e.g.,        with a KD that is less than about 100 pM as determined using a        Biacore affinity assay;    -   i. an IC50 for inhibiting TGFβ transactivation that is about 100        pM to about 300 pM;    -   j. an EC30 for U251 cells of about 100 pM to about 400 pM;    -   k. an EC50 for C8-S cells of about 110 pM to about 180 pM; and    -   l. at least one predicted human pharmacokinetic (PK) parameter        selected from:        -   i. a clearance from central compartment (CL) of about 0.12            mL/h/kg;        -   ii. an inter-compartmental distribution clearance (CLF) of            about 0.51 mL/h/kg;        -   iii. a volume of distribution for the central compartment            (V1) of about 36 mL/kg;        -   iv. a volume of distribution for the peripheral compartment            (V2) of about 33 mL/kg;        -   v. a terminal half-life (t_(1/2)) of about 15 to 17 days;            and        -   vi. no detectable binding to human Fcγ receptors or C1q.            E252. The isolated antibody or antigen-binding fragment            thereof, of any of the preceding embodiments, comprising a            human IgG1 Fc region comprising one or more substitutions            selected from positions L234, L235, and G237 (e.g., one or            more of L234A, L235A, and G237A), as numbered according to            the Eu numbering of Kabat.            E253. The isolated antibody or antigen-binding fragment            thereof, of any of the preceding embodiments, wherein the            antibody is a humanized antibody, a human antibody, a murine            antibody, chimeric antibody, or a camelid antibody.            E254. The isolated antibody or antigen-binding fragment            thereof, of any of the preceding embodiments, wherein the            antibody heavy chain isotype is selected from IgG1, IgG2,            IgG3, IgG4, or any variant thereof; and/or wherein the light            chain constant region is chosen from kappa or lambda.            E255. The isolated antibody or antigen-binding fragment            thereof, of any of the preceding embodiments, wherein the            antibody heavy chain isotype is IgG1 and/or wherein the            light chain constant region is a kappa light chain.            E256. An antibody, or antigen binding fragment thereof, that            competes for binding to αvβ8 integrin with an antibody, or            antigen-binding fragment thereof, of embodiment E242.            E257. A pharmaceutical composition comprising the antibody            or antigen-binding fragment thereof, of any of the preceding            embodiments, and a pharmaceutically acceptable carrier or            excipient.            E258. The pharmaceutical composition of embodiment E257,            comprising i) an antibody or antigen-binding fragment            thereof comprising an antibody heavy chain encoded by the            amino acid sequence of SEQ ID NO:2 and an antibody light            chain encoded by the amino acid sequence of SEQ ID NO:5, ii)            an antibody or antigen-binding fragment thereof comprising            an antibody heavy chain encoded by the amino acid sequence            of SEQ ID NO:3 and an antibody light chain encoded by the            amino acid sequence of SEQ ID NO:5, or iii) both.            E259. An isolated nucleic acid molecule that encodes the            antibody or antigen-binding fragment thereof of any of            embodiments E239-E256.            E260. The isolated nucleic acid of embodiment E259, wherein            the isolated nucleic acid encodes the VH region, VL region,            or both, of the antibody, or antigen-binding fragment            thereof, and wherein said nucleic acid comprises: the            nucleic acid sequence of SEQ ID NO:190, the nucleic acid            sequence of SEQ ID NO:186, or both.            E261. The isolated nucleic acid of embodiment E259, wherein            the isolated nucleic acid encodes the heavy chain constant            region, the light chain constant region, or both, of the            antibody, or antigen-binding fragment thereof, and wherein            the nucleic acid comprises the nucleic acid sequence of SEQ            ID NO: 192 or 193; the nucleic acid sequence of SEQ ID NO:            194; or both.            E262. The isolated nucleic acid of embodiment E259, wherein            the isolated nucleic acid encodes the HC, LC, or both, of            the antibody or antigen-binding fragment thereof, and            wherein said nucleic acid comprises: the nucleic acid            sequence of SEQ ID NO:189 or 190; the nucleic acid sequence            of SEQ ID NO185; or both.            E263. The isolated nucleic acid of embodiment E259, wherein            the isolated nucleic comprises the nucleic acid sequence of            the insert of the plasmid deposited with the ATCC and having            the Accession Number PTA-124917, the nucleic acid sequence            of the insert of the plasmid deposited with the ATCC and            having the Accession Number PTA-124918, or both.            E264. The isolated nucleic acid of embodiment E259, wherein            the isolated nucleic acid comprises a nucleic acid sequence            having at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%,            or 100% sequence identity to SEQ ID NO: 189 or SEQ ID NO:            191; a nucleic acid sequence having at least 80%, 85%, 87%            90%, 92%, 93%, 95%, 97%, 98%, or 100% sequence identity to            SEQ ID NO: 185; or both.            E265. A vector comprising the nucleic acid of any of            embodiments E259-E264.            E266. A host cell comprising the nucleic acid of any of            embodiments E259-E264 or the vector of embodiment E265.            E267. The host cell of embodiment E265, wherein the host            cell is a mammalian cell selected from the group consisting            of a CHO cell, a COS cell, a HEK-293 cell, an NS0 cell, a            PER.C6® cell, and an Sp2.0 cell.            E268. A method of making an isolated antibody, or            antigen-binding fragment thereof, comprising culturing the            host cell of embodiment 266, under conditions wherein the            antibody or fragment is expressed by the host cell and            isolating the antibody or fragment.            E269. A method of reducing αvβ8 integrin activity in a            subject in need thereof, the method comprising administering            to the subject a therapeutically effective amount of the            antibody, or antigen-binding fragment thereof, of any of            embodiments E239-E256, or the pharmaceutical composition of            embodiments E257 or E258.            E270. A method of treating cancer, comprising administering            to a subject in need thereof, a therapeutically effective            amount of the antibody or antigen-binding fragment thereof            of any of embodiments E239-E256, or the pharmaceutical            composition of embodiments E257 or E258.            E271. The method of embodiment E270, further administration            of a cytotoxic agent, a cytostatic agent, a chemotherapeutic            agent, a hormone treatment, a vaccine, an immunotherapy,            surgery, radiation, cryosurgery, thermotherapy, or a            combination thereof.            E272. The method of embodiment E271, wherein the further            administration is simultaneous, sequential or separate from            the administration of the therapeutically effective amount            of the antibody, or antigen-binding fragment thereof, or the            pharmaceutical combination.            E273. The method of embodiment E271, wherein the            immunotherapy comprises a modulator of an immune checkpoint            molecule selected from the group consisting of an anti-PD1            antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an            anti-CTLA-4 antibody, a soluble CTLA-4 fusion protein and a            combination thereof, and wherein the anti-PD-L1 antibody is            not avelumab.            E274. The method of any one of embodiments E270-E273,            wherein the cancer is selected from the group consisting of            squamous cell carcinoma of the head and neck, renal cell            carcinoma with clear cell or papillary cell type, ovarian            cancer, fallopian tube cancer, primary peritoneal cancer,            gastric cancer, gastroesophageal junction cancer, esophageal            cancer, lung squamous cell cancer, pancreatic ductal            adenocarcinoma, cholangiocarcinoma, uterine cancer,            melanoma, urothelial carcinoma and combinations thereof.            E275. A method of detecting αvβ8 integrin in a sample,            tissue or cell using the antibody, or antigen-binding            fragment thereof, of embodiments E239-E256, comprising            contacting the sample, tissue or cell with the antibody, or            antigen-binding fragment thereof, and detecting the            antibody, or antigen-binding fragment thereof.            E276. A kit comprising the antibody or fragment of any of            embodiments E239-E256, or the pharmaceutical composition of            embodiments E257 or E258 and optionally comprising the            modulator of embodiment E273.            E277. The antibody or antigen-binding fragment thereof            according to any of embodiments E239-E256, or the            pharmaceutical composition of embodiments E257 or E258 for            use in reducing αvβ8 integrin activity in a subject in need            thereof, for treatment of cancer.            E278. The antibody, or antigen-binding fragment thereof, of            any of embodiments E239-E256, or the pharmaceutical            composition of embodiments E257 or E258 for use in treating            cancer, optionally wherein the antibody, or antigen-binding            fragment thereof, or the pharmaceutical composition is for            administration simultaneously, sequentially or separately in            combination with immunotherapy wherein the combination            optionally provides a synergistic therapeutic effect.            E279. The antibody or antigen-binding fragment thereof, or            the pharmaceutical composition for use according to            embodiment E278, wherein the cancer is selected from the            group consisting of squamous cell carcinoma of the head and            neck, renal cell carcinoma with clear cell or papillary cell            type, ovarian cancer, fallopian tube cancer, primary            peritoneal cancer, gastric cancer, gastroesophageal junction            cancer, esophageal cancer, lung squamous cell cancer,            pancreatic ductal adenocarcinoma, cholangiocarcinoma,            uterine cancer, melanoma, urothelial carcinoma and            combinations thereof, optionally wherein the antibody, or            antigen-binding fragment thereof, or the pharmaceutical            composition or combination are for use together with            administration of immunotherapy or radiation therapy.            E280. Use of an antibody, or antigen-binding fragment            thereof, of any one of embodiments E239-E256, or the            pharmaceutical composition of embodiments E257 or E258 for            treating cancer.            E281. Use of an antibody, or antigen-binding fragment            thereof, of any one of embodiments E239-E256 in the            manufacture of a medicament for treating cancer.            E282. A method of treating cancer, comprising administering            to a subject in need thereof, a therapeutically effective            amount of (i) an antibody or antigen-binding fragment            thereof that specifically binds αvβ8 integrin and (ii) a            modulator of an anti-PD1, anti-PD-L1 or anti-PD-L2 immune            checkpoint molecule.            E283. The method of embodiment E282, wherein the cancer is a            squamous cell carcinoma.            E284. The method of embodiment E282, wherein the cancer is            breast or colon cancer.            E285. The method of any one of embodiments E282-E284,            wherein the modulator is selected from the group consisting            of an anti-PD1 antibody, an anti-PD-L1 antibody, and an            anti-PD-L2 antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention thefollowing drawings embodiment(s) are shown, however, it should beunderstood that the invention is not limited to the precise arrangementsand instrumentalities shown.

FIG. 1A shows a sequence alignment comparing the heavy chain variableregion amino acid sequences of the mouse hybridoma antibody ADWA11(referred to as “mADWA11” “ADWA11” or “Hybridoma mouse ADWA11”; SEQ IDNO: 20), the humanized ADWA11 VH05-2/VK01(2.4) antibody (“huADWA11-2.4”,“ADWA11 2.4” or “humanized ADWA11-2.4”; SEQ ID NO: 6), and the IGHV3-07germline (“IMGT” or “DP-54”; SEQ ID NO: 195) sequences. The underlinedamino acid residues are the CDR sequences according to Kabat.

FIG. 1B shows a sequence alignment comparing the light chain variableregion amino acid sequences of the mouse hybridoma antibody ADWA11(referred to as “mADWA11” “AWDA11” or “Hybridoma mouse ADWA11”; SEQ IDNO: 21), the humanized ADWA11 VH05-2/VK01(2.4) antibody (“huADWA11-2.4”,“ADWA11 2.4” or “humanized ADWA11 2.4”; SEQ ID NO: 7), and the IGKV1-39germline (“IMGT” or “DPK-9”; SEQ ID NO: 196) sequences. The underlinedamino acid residues are the CDR sequences according to Kabat.

FIG. 2 shows a representative graph comparing the binding specificity ofthe mouse hybridoma antibodies ADWA2 and ADWA11 for human integrins αvβ3(“AVB3”) and αvβ6 (“AVB6”), as determined by ELISA. ADWA2 and ADWA11 didnot bind integrins αvβ3 (“AVB3”) and αvβ6 (“AVB6”), while the control αVbinding antibody (“AlphaV mAb”) bound both αvβ3 and αvβ6.

FIG. 3A shows a representative graph showing that the mouse hybridomaADWA11 antibody bound to human αvβ8 as determined by ELISA.

FIG. 3B shows a representative graph showing that the humanized antibodyADWA11 VH05/VK01(2.4) bound to human αvβ8 as determined by ELISA.

FIG. 4A shows a representative graph showing the binding affinity, asdetermined by ELISA, of ADWA11 VH05/VK01 Fabs having a K30A, N55Q, N57Q,D61E, P62A, or K63A amino acid substitution in the heavy chain variableregion, to human αvβ8, as compared to the binding affinity of parentalhumanized ADWA11_VH05/VK01 (ADWA VH_1.5 and ADWA VL_1.1) antibody tohuman αvβ8. Fabs having a K30A, N55Q, N57Q, D61E, P62A, or K63A aminoacid substitution in the heavy chain variable region retained bindingaffinity for human αvβ8.

FIG. 4B shows a representative graph showing the binding affinity, asdetermined by ELISA, of ADWA11 VH05-2/VK01 Fabs having an amino acidsubstitution in either the heavy chain variable region (e.g., F64V), orthe light chain variable region (e.g., L30S, Y55A, A60Q, M94Q, L97Y,F101L, F101W, or Q105G), to human αvβ8, as compared to the bindingaffinity of parental ADWA11 VH05-2/VK01 antibody to human αvβ8. Fabshaving a Y55A, A60Q, F101L, or F101W amino acid substitution in thelight chain variable region displayed a reduced binding affinity tohuman αvβ8, as compared to the humanized ADWA11 VH05-2/VK01 antibody.The other tested Fabs retained binding affinity for human αvβ8.

FIG. 4C shows representative graphs showing the binding affinity, asdetermined by ELISA, of ADWA11 VH05-2/VK01 Fabs referred to asVH05-2/VK01(2.1) (ADWA11 2.1), VH05-2/VK01(2.2) (ADWA 2.2),VH05-2/VK01(2.3) (ADWA 2.3), VH05-2/VK01(2.4) (ADWA 2.4),VH05-2(F64V)/VK01(2.1), VH05-2(F64V)/VK01(2.3), andVH05-2(F64V)/VK01(2.4), having a combination of amino acid substitutionsas shown in Table 5, to human αvβ8, and as compared to the parentalantibody (VH05-2 VK01 parental). Each of the tested Fabs retainedbinding affinity for human αvβ8.

FIG. 5 shows a representative graph comparing the binding specificity ofthe mouse hybridoma ADWA11 (“MsADWA11” or “mADWA11”) and humanizedADWA11 VH05-1/VK01 (“ADWA11 5-1_1”) and ADWA11 VH05-2/VK01 (“ADWA115-2_1”) for human integrins αvβ3 (αvb3) and αvβ6 (αvb6), as determinedby Biacore. The ADWA11 antibodies did not bind integrins αvβ3 (αvb3) andαvβ6 (αvb6), while the control αV binding antibody (“anti-αv”) boundboth αvβ3 and αvβ6.

FIG. 6A shows a representative Biacore binding trace for the hybridomaADWA11 (“Ms ADWA11”) Fab to human αvβ8.

FIG. 6B shows a representative Biacore binding trace for the humanizedADWA11_5-2 2.4 Fab, also referred to as ADWA11 VH05-2/VK01(2.4) or“ADWA11 2.4” Fab to human αvβ8.

FIG. 6C shows a representative table showing that the humanized FabADWA11, referred to herein as ADWA11 5-2 2.4 (also known as ADWA11 2.4,and ADWA11 VH05-2/VK01(2.4)), retains affinity for human αvβ8 and crossspecies reactivity as assessed by Biacore, as compared to the parentalmouse antibody Fab (“MsADWA11”). ADWA11 5-2 2.4 demonstrated anequivalent affinity for human, cynomolgus, mouse, and rat αvβ8 with a KDof <200 pM. The parental mouse antibody demonstrated an equivalentaffinity for human, cynomolgus, and mouse αvβ8 with a KD of 489-536 pM.

FIG. 7A shows a representative graph comparing U251 cell binding datafor ADWA11 VH05-2/VK01 (Parental) Fabs (“ADWA11”) having a single aminoacid substitution in either the heavy chain variable region (e.g.,F64V), or the light chain variable region (e.g., L30S, M94Q, L97Y,F101L, or Q105G). Fabs having a F101L amino acid substitution in thelight chain variable region displayed reduced binding to U251 cells(human αvβ8), as compared to the parental antibody. The other testedFabs retained binding to U251 cells (human αvβ8).

FIG. 7B shows a representative graph comparing U251 cell binding datafor ADWA11 VH05-2/VK01 (Parental) Fabs (“ADWA11”) having a single aminoacid substitution in the light chain variable region (e.g., M56A orN58S). The M56A and N58S Fabs retained binding to U251 cells (humanαvβ8).

FIG. 7C shows a representative graph showing U251 cell binding data forADWA11 VH05-2/VK01 Fabs (“ADWA11”) having a combination of amino acidsubstitutions referred to as 2.1, 2.2, 2.3, 2.4, 2.1 (F64V), 2.3 (F64V),and 2.4 (F64V) according to Table 5, as compared to the parentalantibody. Each of the tested Fabs retained binding to fixed U251 cells.

FIG. 8 shows a representative graph comparing the binding affinities ofantibodies ADWA11 mIgG_4mut and ADWA11 VH05/VK01 to U251 cells.

FIG. 9A shows representative graphs showing binding of ADWA11VH05-2/VK01 having a combination of amino acid substitutions referred toas ADWA11_VH05-2/VK01(2.1), and ADWA11_VH05-2/VK01(2.4) to U251 (humanglioblastoma) or C8-S (mouse astrocyte) cells. The ADWA11VH05-2/VK01(2.1) and ADWA11 VH05-2/VK01(2.4) antibodies retained bindingto U251 cells (human αvb8) and C8-S (mouse αvb8).

FIG. 9B shows representative graphs depicting binding of integrinspecific antibodies, such as, ADWA11 VH05-2NK01(2.4) to HEK cellsexpressing αvβ3, αvβ5, αvβ6, and αvβ8. Results show saturable binding ofADWA11 VH05-2/VK01(2.4) to HEK cells expressing αvβ8 and no binding tocells expressing αvβ3, αvβ5, αvβ6. There results demonstrate specificbinding of ADWA11 VH05-2/VK01(2.4) to human αvβ8.

FIG. 10A shows a representative graph comparing the effect of mousehybridoma ADWA11 (“mFab”) and humanized ADWA11 Fabs: ADWA11 VH01/VK01,ADWA11 VH02/VK01, ADWA11 VH02/VK02, ADWA11 VH05/VK02, and ADWA11VH05/VK01 on TGFβ trans-activation by U251 cells. The VH01/VK01,VH02/VK01, and VH02/VK02 Fabs displayed reduced activity, whileVH05/VK01 retained activity, and VH05/VK02 demonstrated improvedactivity to block TGFβ activation in the U251 transactivation assay ascompared to mouse hybridoma ADWA11 Fab (“mFab”).

FIG. 10B shows a representative graph comparing the effect of theindicated ADWA11 VH05-2/VK01 Fabs having an amino acid substitution ineither the heavy chain variable region (e.g., F64V), or the light chainvariable region (e.g., L30S, M94Q, L97Y, F101L, F101W, or Q105G) on TGFβtransactivation in U251 cells. Fabs having a F101L or F101W single aminoacid substitution in the light chain variable region displayed a reducedeffect on TGFβ transactivation, as compared to the humanized parentalADWA11 VH05-2/VK01 Fab. The other tested Fabs retained activity in theTGFβ transactivation assay.

FIG. 10C shows a representative graph comparing the effect of ADWA11VH05-2/VK01 Fabs having amino acid substitutions, including thecombination of amino acid substitutions referred to as 2.1, 2.2, 2.3,2.4, 2.1 (F64V), 2.3 (F64V), and 2.4 (F64V) according to Table 5. TheVH02-2/VK01(2.3) and VH05-2(F64V)/VK01(2.3) Fabs displayed a reducedeffect on TGFβ transactivation, as compared to the parental ADWA11VH05-2/VK01 Fab. The other tested Fabs retained activity in the TGFβtransactivation assay.

FIG. 10D shows a representative graph comparing the effect of ADWA11VH05-2/VK01 (Parental) Fabs having the indicated amino acid substitutionin the heavy chain variable region (e.g., F64V), or the light chainvariable region (e.g., L30S, M94Q, L97Y, Q105G, M56A, or N58S). Thetested Fabs retained activity in the TGFβ transactivation assay ascompared to the parental ADWA11 VH05-2/VK01 Fab.

FIG. 10E shows a representative graph showing the effect of thehumanized ADWA11 VH05/VK01, VH05/VK01-D61E (VH05-1/VK01), andVH05/VK01-N55Q-D61E (VH05-2/VK01) IgG on TGFβ transactivation by U251cells. The tested antibodies retained activity in the TGFβtransactivation assay.

FIG. 10F depicts representative graphs showing the effect ofADWA11_VH05-2_VK01(2.4) on TGFβ transactivation by U251 cells (leftpanel) and C8-S (right panel), compared to the isotype control antibody.Additional experiments demonstrated the IC50 for ADWA11 VH05-2/VK01(2.4)in the TGFβ transactivation assay with U251 cells to be 199±93.6 pM(average±standard deviation).

FIG. 11 shows a representative graph showing the percentage ofresponders (antigenicity) for different ADWA11 VH05-2VK01 CDR peptidescompared to positive control peptides set forth in Table 1. Peptideantigenicity score was used to select possible CDR sequences withreduced immunogenicity risk.

FIG. 12A shows representative graphs showing the efficacy ofcombinations of anti-αvβ8 (ADWA11), anti-PD1 antibody (“PD-1”, RMP1-14),mIgG1_4mut isotype (2B8), and rat IgG2a isotype (2A3) treatment in theEMT6 breast cancer tumor model. Tumor growth was measured using digitalcalipers three times per week and reported as tumor volume(length×width×width×0.5). Mean tumor volume+/−SEM in each treatmentgroup was plotted until less than 8/10 of mice were remaining in eachgroup. Survival was defined as the time to reach 1000 mm³. Thecombination of anti-PD1 and ADWA11 inhibited tumor growth and improvedoverall survival to a greater extent than the other combinations tested.

FIG. 12B shows representative graphs showing the efficacy of ananti-αvβ8 antibody (ADWA11) at 1, 3, 10, and 20 mg/kg as monotherapy andan isotype control (mIgG1_4mut isotype (2B8)) in the EMT6 breast cancermodel in the top panel. Also shown is the combination of anti-αvβ8antibody ADWA11 at 1, 3, 10, and 20 mg/kg with anti-PD1 antibody(RMP1-14, 10 mg/kg) and a rat IgG2a isotype (2A3) in the EMT6 breastcancer tumor model. Mice were treated with antibodies on Day 0, 4, and 8of the study and tumor growth was measured using digital calipers threetimes per week and reported as tumor volume (length×width×width×0.5).

FIG. 13 shows representative graphs showing the efficacy of combinationsof anti-αvβ8 (ADWA11), anti-41BB (MAB9371), anti-CTL4 (9D9), mIgG1_4mutisotype (2B8), and rat IgG2a isotype (2A3) treatment in an EMT6 tumormodel. Tumor growth was measured using digital calipers three times perweek and reported as tumor volume (length×width×width×0.5), mean tumorvolume+/−SEM in each treatment group was plotted until less than 8/10 ofmice were remaining in each group and survival was defined as the timeto reach 1000 mm³. The combination of anti-4-1BB and ADWA11 oranti-CLTA4 and ADWA11 treatment inhibited tumor growth and improvedoverall survival to a greater extent than the other combinations tested.

FIG. 14A depicts representative graphs showing that the combination ofanti-αvβ8 antibody and radiation therapy (ADWA11+5Gy radiation group)inhibited tumor growth and improved overall survival to a greater extentthan radiation therapy with an isotype control (mIgG4mut (2B8)+5Gyradiation group) in the CT26 tumor model. Tumor growth was measuredusing digital calipers 3 times per week and reported as tumor volume(length×width×width×0.5), mean tumor volume+/−SEM in each treatmentgroup is plotted and survival was defined as the time to reach 1000mm³. * p<0.05 vs No Treatment, ** P<0.05 vs radiation+2B8.

FIG. 14B top graph depicts the density of CD45, CD8, and Granzyme Bexpressing cells in CT26 tumor tissue collected on Day 12 from micetreated on Day 0, Day 4, and Day 8 with 10 mg/kg of isotype control(Control), or ADWA11 antibody (Anti-ITGαVβ8), n=6; p=P-value. The bottomgraph depits gene expression of CD45, CD8, GranzymeB, and IFNγ in tumortissue collected 12 days after the first 10 mg/kg dose of isotypecontrol (Isotype), ADWA11 antibody (Anti-ITGαVβ8), Isotype incombination with 5 Gy of tumor targeted radiation, or ADWA11 antibody incombination with 5Gy of tumor targeted radiation. Antibody treatmentswere administered intravenously on Day 1, Day 4, and Day 8 of the studyand radiation therapy was administered on Day 5 of the study. Five micewere included in each treatment group; mean and standard error of themean are graphed.

FIG. 15 depicts representative graphs showing IHC analysis of thedensity of CD45 (total lymphocytes and myeloid cells), CD3 (total Tcells), CD4 T cells, CD8 T cells, and Granzyme B (activated CD8 and NKcells) in the EMT6 tumor model. ADWA11(2.4) (also referred to asADWA11VH05-2/VK01(2.4) herein) treatment increased the density of allcell types analyzed. N=10 per group, p value for two-tailed t-testlabelled on graph.

FIG. 16A shows a diagram showing a treatment regimen for the CCK168tumor model. The time line for implantation of tumor cells andintraperitoneal (i.p.) antibody injection for four treatment groups isprovided.

FIG. 16B shows representative Kaplan-Meier survival curves using a tumorvolume of 2000 mm³ as a cutoff for survival in the CCK168 tumor model.n=10 in each group. **p<0.01 by log-rank Mantel-Cox test.

FIG. 16C depicts representative graphs showing individual growth curvesof tumors shown in FIG. 16B. Mice were euthanized prior to the 45-dayendpoint when tumors reached≥2000 mm³ or if extensive tumor ulcerationwas observed. The combination of an anti-PD1 antibody and ADWA11treatment synergistically inhibited tumor growth and improved overallsurvival to a greater extent than an anti-PD1 antibody, ADWA11, orisotype control treatment alone or the individual effects merely addedtogether. Data shown are representative of 3 independent biologicalreplicates.

FIG. 17A depicts the gating strategy for identification of tumorinfiltrating monocytes, macrophages, and dendritic cells. Live singlecells were first gated with a dump gate including Ly6G, SiglecF, CD90and B220 to eliminate neutrophils, eosinophils, lymphocytes and B cells.Negatively staining cells were then analyzed by flow cytometry andgated. Macrophages were identified as CD45+CD11b+CD64^(high)F4/80^(high)and dendritic cells were identified asCD45+CD11b+F4/80-CD64-MHCII^(high)CD11c^(high). Of the macrophagepopulation, profiles consistent with immunostimulatory macrophages wereidentified as Ly6C^(high)CD206^(low) and immunosuppressive macrophageswere identified as Ly6C^(low)CD206^(high). Dump channelLy6G+SiglecF+CD90.2+B220+.

FIG. 17B shows a representative graph showing cell surface staining forintegrin αvβ8 analyzed as part of a multicolor flow cytometry panel indisaggregated tumors described in FIG. 17A. Representative flowcytometry plots showing fluorescence minus one control staining (FMO)and ADWA11 antibody staining in CCK168 tumor model. N=4 tumors.

FIG. 18A depicts representative graphs showing ADWA11 staining onCD45-negative cells isolated from CCK168. Live single cells wereanalyzed for the presence of CD45. 4% of CD45 negative cells in theCCK168 tumor model were positive for αvβ8 expression using ADWA11antibody.

FIG. 18B depicts the expression of αvβ8 in the CCK168, CT26, and EMT6cell lines. Live single cells were analyzed for αvβ8expression by flowcytometry using isotype and anti-αvβ8 (ALDWA11) staining antibodies. TheCCK168 and EMT6 cell lines have detectable expression of αvβ8, while theCT26 cells line does not.

FIG. 19A depicts representative graphs showing total intratumoral CD8+ Tcells numbers of mice with CCK168 tumors treated with control antibodiesalone, anti-PD1, ADWA11 or combined anti-PD1 andADWAll. Representativeflow cytometry plots for CD4 and CD8 expression of all CD45+ cells areshown along with the ratio of CD8+ cells number to CD45+ cells for eachmouse in each group.

FIG. 19B shows representative immunofluorescence micrographs of CCK168tumor sections harvested from mice in each treatment group, stained foranti-CD8 and DAPI. Scale bar, 50 μm.

FIG. 19C shows representative flow cytometry plots showing intracellularstaining for Granzyme B in cells gated for CD8 expression, along withthe percentage of CD8+ cells expressing detectable Granzyme B in eachmouse in each group.

FIG. 19D shows representative flow cytometry plots showingimmunostimulatory macrophages (Ly6c^(high), CD206^(low)), andimmunosuppressive macrophages (CD206^(high), Lytic^(low)) for cellsgated as CD45+Ly6G-CD11b+CD64^(high)F4/80^(high). Of the macrophagepopulation, immunostimulatory macrophages were identified asLy6C^(high)CD206^(low) and immunosuppressive macrophages were identifiedas Ly6C^(low)CD206^(high), along with percentage of immunostimulatorymacrophages for each mouse. Data in graphs are mean±SEM, n=10 per group.*p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA.

FIG. 20 depicts representative graphs showing ADWA11 staining on CD4+and CD8+ T cells in the CCK168 tumor microenvironment. Live, singleCD45+ cells were gated on CD4 and CD8 and stained with ADWA11.Representative plots are shown for mice from each of the 4 treatmentgroups.

FIG. 21A shows representative graphs showing immuno-depletion of CD8+ Tcells. Micrographs show immunostaining with anti-CD8 counterstained withDAPI in CCK168 tumors isolated following combinatorial anti-PD-1/ADWA11therapy with or without prior treatment with anti-CD8 depleting antibodyor isotype control antibody.

FIG. 21B shows representative graphs showing average tumor growth curvesfor CCK168 tumors pretreated with anti-CD8 depleting antibody or isotypecontrol antibody 24 hours prior to ADWA11/anti-PD-1 combination therapy.

FIG. 21C shows representative graphs showing survival of mice harboringCCK168 tumors following ADWA11/anti-PD-1 combination therapy, pretreatedone day earlier with either anti-CD8 depleting antibody or isotypecontrol antibody. Data reported as percent survival, n=10 in each group.**p<0.01 by log-rank Mantel-Cox test.

FIG. 22A shows representative graphs showing CCK168 tumors harvestedfrom mice treated with either ADWA-11 or control antibody, and stainedwith antibodies to, CD8, F4/80 to detect macrophages and phospho-SMAD3to detect TGFβ signaling (pS3). Low power merged images and imagesshowing only pSMAD3 are shown to the left and enlarged merged images andimages for each single antibody from boxed areas are shown to the right.

FIG. 22B shows representative graphs depicting quantification of pSmad3(pS3) staining density in control and ADWA11 treated mice. ADWA11treatment decreased pSmad3 density in CCK168 tumors.

FIG. 23A shows representative graphs setting forth data extracted fromThe Cancer Genome Atlas (TCGA) for integrin-β8 mRNA expression in 30different human cancers. Each dot represents an individual tumor sample.The results shown in this figure are based upon data generated by theTCGA Research Network.

FIG. 23B shows representative graphs showing flow cytometry data ofdisaggregated cells from fresh, de-identified ovarian carcinoma andrenal cell carcinomas gated for mature monocytes (CD16+ monocytes),tumor associated macrophages (CD14+ macrophages), two monocyte deriveddendritic cell populations (BCDA1+ moDCs and BCDA3+ moDCs) oreosinophils and stained for expression of αvβ8. Tissues from normaltonsils were also analyzed as a control. Gating strategy is shown inFIG. 23C; n=2 for each tumor type.

FIG. 23C are representative graphs showing the gating strategy for humantumor biopsy samples. Live single CD45+ cells were gated for SSC-A(hi)to remove granulocytes. SSC-A(lo) was gated for HLA-DR+CD3- and stainedwith CD14 and CD16. CD14+CD16+ were designated CD16+ monocytes. CD16−cells were further stained for CD11c. CD14+CD11c+ cells were furtherstained with CD1c and CD141. CD1c+CD141− were designated CD1c+ MoDC.CD141+CD1c− were designated CD141+ MoDC. CD1c-CD141− cells were furtherstained with CD64 and CD64+ population were designated CD14+TAMs.

FIG. 24A shows a representative graph showing the results of a TMLC cellco-culture bioassays performed with concentrations of ADWA11 rangingfrom 0.01 to 10 mg/ml. TGFβ activity is reported as relative luciferaseunits based on PAI-1 luciferase reporter activity. n=3 per ADWA11 dose,repeated 3 times.

FIG. 24B shows a representative graph showing the results of a celladhesion assays performed on dishes coated with the latency associatedpeptide (LAP) of TGFβ1 in the presence of ADWA-11 in concentrations from0.001 to 10 mg/ml. Adherent cells were stained with crystal violet andadhesion expressed as absorbance at 595 nm. n=3 per ADWA11 dose,repeated 3 times.

FIG. 24C depicts representative graphs showing that antibodies tointegrin αvβ3, αvβ5, αvβ6, and αvβ8 were used for flow cytometry of wildtype colon carcinoma cells, SW-480, that do not express any of theseintegrins or SW-480 cells transfected to express (33 (SW-itgb3), (36(SW-itgb6), or (38 (SW-itgb8). Representative flow cytometry plots areshown for each antibody and cell type tested.

FIG. 25A shows a representative graph depicting two-compartmentnon-linear pharmacokinetic model fitted to 0.1, 0.3, and 3 mg/kg i.v.dosing of ADWA11(2.4) in TG32 mice Circles: observed data. Lines: modelfit.

FIG. 25B shows a representative graph depicting two-compartmentpharmacokinetic model fitted to 3 mg/kg i.v. dosing of ADWA11 2.4 inTG32 mice. Circles: observed data. Lines: model fit.

FIG. 26 shows representative graphs depicting two-compartmentpharmacokinetics of ADWA11 2.4 in cynomolgus monkeys following a singleIV bolus administration at 4, 40, and 100 mg/kg, respectively. Circles:observed data. Lines: model fit.

FIGS. 27A-27B show representative graphs illustrating predicted humanADWA11 2.4 pharmacokinetics following 12 mg/kg Q28D and 7 mg/kg Q14D. CPis the predicted plasma concentration; CAVG is the Cavg or averageplasma concentration; Rodent NOAEL is the C_(ave) no observed adverseeffect level in rodents; and NHP NOAEL is the C_(ave) no observedadverse effect level in nonhuman primates.

FIG. 28 depicts representative graphs showing Kaplan-Meier survivalcurves, using a tumor volume of 2000 mm³ as a cutoff for survival forthe CCK168 tumor model. Treatment groups were isotype control, anti-PD1antibody, ADWA11_4mut, and a combination of anti-PD1 antibody andADWA11_4mut. A combination of anti-PD1 antibody and ADWA11 4muttreatment synergistically improved overall survival to a greater extentthan anti-PD1 antibody, ADWA11_4mut, or isotype control treatment aloneor the expected additive effects of combination treatments.

FIG. 29A shows graphs depicting representative survival curves and FIG.29B shows representative individual tumor growth curves in miceimplanted with subcutaneous CT26 cells and treated with isotype controlantibodies, anti-PD1, ADWA11_4mut, or a combination of anti-PD1 andADWA11_4mut, plus 5 Gy radiation dose on day 5. One group of micetreated with isotype control antibody did not receive radiation therapy.Data reported as percent survival, n=10 in each group. *** p<0.001,****p<0.0001 by log-rank Mantel-Cox test.

FIG. 29C shows graphs depicting representative tumor re-challenge inCT26-cured mice that survived 50 days post-treatment initiation.Parental CT26 cells were implanted into the flank contralateral to thatof the original tumor implantation site of CT-26-cured mice, 51 daysafter initiating immunotherapy in combination with radiation therapy.Control mice did not receive radiation and were not previously exposedto tumor cells. Re-challenged mice were followed for 30 days. Control,n=10; RT plus anti-PD1, n=3; RT plus ADWA11_mut, n=5; RT plus ADWA11_mutand anti-PD1, n=7.

FIG. 30A shows graphs depicting representative survival curves and FIG.30B shows graphs depicting representative individual growth curves inmice implanted orthotopically with EMT6 cells following treatment withisotype control antibody, ADWA11_4mut, 4-1BB, anti-CTLA4, anti-PD-1, ora combination of ADWA-11_4mut and 4-1BB, anti-CTLA4 or anti-PD-1. Datareported as percent survival, n=10 in each group. **** p<0.0001 bylog-rank Mantel-Cox test

FIG. 30C shows graphs depicting representative survival curves and FIG.30D shows graphs depicting representative individual growth curves formice treated with anti-CTLA4 or activator of 4-1BB.

FIG. 30E shows representative graphs depicting results from tumorre-challenge in mice that survived 50 days with complete regression oftumors after treatment with the synergistic combination of ADWA-11_4mutand anti-CTLA4 or the synergistic combination of ADWA-11_4mut andactivator of 4-1BB. Control mice were not previously exposed to tumor.Re-challenged mice were assessed for 30 days. n=5 control, n=6ADWA-11_4mut+anti-CTLA4, n=5 ADWA-11_4mut+4-1BB.

FIG. 31A depicts mRNA gene expression analysis in MC38 tumor tissueusing CD8a and GranzymeB specific taqman probes. Tumor tissue wascollected 12 days after the first dose of Isotype control or ADWA11 2.4antibody at the indicated dosage level. Treatments were administeredintravenously on Day 1, Day 5, and Day 9 of the study, 5 mice wereincluded in each treatment group. *=p-value<0.05.

FIG. 31B shows representative graphs depicting tumor growth rate in theMC38 tumor model in Isotype (10 mg/kg), ADWA11 2.4 (10 mg/kg), anti-PD-1antibody (RMP1-14, 10 mg/kg), and combined ADWA11 2.4 (10 mg/kg) andanti-PD1 antibody (RMP1-14, 10 mg/kg) treated mice. Additionally,representative graphs showing the tumor growth rate of MC38 tumors inmice treated with ADWA11 (anti-ITGAVB8 (ADWA11_mIgG_4mut) antibody at a0.03, 0.3, 3, and 30 mg/kg dose in combination with 10 mg/kg ofanti-PD-1 antibody (RMP1-14). For all graphs antibodies wereadministered on Day 1, 5, and 9 of the study.

DETAILED DESCRIPTION

Disclosed herein are antibodies, and antigen-binding fragments thereof,that specifically bind to αvβ8 integrin (e.g., human αvβ8 integrin) andfurther, antibodies that antagonize αvβ8 integrin activity (e.g.,antagonizes activation of TGFβ, antagonizes mediation of TGFβproduction, antagonizes modulation of Tregs and Th17 cells) or itsinteraction with TGFβ1 and TGFβ3, or the release of active TGFβ. Methodsof making anti-αvβ8 integrin antibodies, compositions comprisinganti-αvβ8 integrin antibodies, and methods of using anti-αvβ8 integrinantibodies are provided. In some embodiments, recombinant, e.g.,humanized, antibodies that bind αvβ8 integrin (e.g., human αvβ8integrin) are provided. In some embodiments, humanized antibody heavychains and light chains that are capable of forming antibodies that bindαvβ8 integrin are also provided. In some embodiments, humanizedantibodies, heavy chains, and light chains comprising one or moreparticular complementarity determining regions (CDRs) are provided. Insome embodiments, humanized anti-αvβ8 integrin antibodies have alteredeffector functions. In some embodiments, the antibodies of the inventionhave reduced antibody-dependent cell-mediated cytotoxicity (ADCC)activity and/or complement dependent cytotoxicity (CDC) activityrelative to otherwise identical anti-αvβ8 integrin antibodies of theinvention.

Polynucleotides encoding antibodies that bind αvβ8 integrin (e.g., humanαvβ8 integrin), or antigen-binding fragments thereof, are provided.Polynucleotides encoding antibody heavy chains or light chains are alsoprovided. Host cells that express anti-αvβ8 integrin antibodies,including humanized antibodies, are provided. Methods of treatment usinganti-αvβ8 integrin antibodies, including humanized antibodies, areprovided.

Anti-αvβ8 integrin antibodies, and antigen-binding fragments thereof,including humanized antibodies, can be used in the prevention,treatment, and/or amelioration of diseases, disorders, or conditionscaused by and/or associated with aberrant (e.g., increased) TGFβsignaling. Such diseases, disorders, or conditions include cancer (e.g.,controlling the proliferation of cancer cells with aberrant (e.g.,increased) TGFβ signaling).

Without wishing to be bound by any particular theory, mature TGFβ ispresent in inactive or latent form in a complex with the latencyassociated peptide (LAP) domain. Binding of αvβ8 integrin to LAP resultsin release of active TGFβ (e.g., TGFβ1 and TGFβ3). Reducing binding ofαvβ8 integrin to LAP can prevent the release of active TGFβ, therebyreducing TGFβ signaling. TGFβ is known to have immune suppressiveeffects, e.g., in the tumor microenvironment, thus reduction of TGFβactivity and/or signaling using the antibodies described herein canresult in activation of an immune response, e.g., an anti-tumor responsein vivo. Thus, antibodies, and antigen-binding fragments thereof, of thedisclosure enable selective antagonism of TGFβ activity in the immunesystem and/or the tumor microenvironment, thus enhancing an anti-tumorimmune response in a subject. In some embodiments disclosed in theExamples herein, antibodies, and antigen-binding fragments thereof,against αvβ8 integrin have been shown to cause growth suppression and/orcomplete tumor regression in animal models for several cancers,including squamous cell carcinoma, breast, and colon cancer, alone or incombination with other immunomodulators, such as modulators ofcheckpoint inhibitors, (e.g., inhibitors of PD-1, PD-L1, CTLA-4 oragonists of 4-1BB), or anti-cancer therapies, e.g., radiotherapy. Thus,anti-αvβ8 integrin antibodies, and antigen-binding fragments thereof,including humanized antibodies, can be used, alone or in combinationwith a second therapy, in the prevention, treatment, and/or ameliorationof a cancer, e.g., a solid tumor, e.g., a solid tumor chosen from: renalcell carcinoma (RCC), an ovarian cancer, or a head and neck squamouscell carcinoma (SCCHN).

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

All references cited herein, including patent applications, patentpublications, and Genbank Accession numbers are herein incorporated byreference, as if each individual reference were specifically andindividually indicated to be incorporated by reference in its entirety.

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J. B. LippincottCompany, 1993); and updated versions thereof.

I. DEFINITIONS

The present invention may be understood more readily by reference to thefollowing detailed description of exemplary embodiments of the inventionand the examples included therein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent specification, including definitions, will control.

Further, unless otherwise required by context or expressly indicated,singular terms shall include pluralities and plural terms shall includethe singular.

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments. As used herein, the singular form “a”, “an”, and “the”includes plural references unless indicated otherwise.

In this application, the use of “or” means “and/or” unless expresslystated or understood by one skilled in the art. In the context of amultiple dependent claim, the use of “or” refers back to more than onepreceding independent or dependent claim.

“About” or “approximately,” when used in connection with a measurablenumerical variable, refers to the indicated value of the variable and toall values of the variable that are within the experimental error of theindicated value (e.g. within the 95% confidence interval for the mean)or within 10 percent of the indicated value, whichever is greater.Numeric ranges are inclusive of the numbers defining the range.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a stated range of “1 to 10” should be consideredto include any and all subranges between (and inclusive of) the minimumvalue of 1 and the maximum value of 10; that is, all subranges beginningwith a minimum value of 1 or more, e.g. 1 to 6.1, and ending with amaximum value of 10 or less, e.g., 5.5 to 10.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers. Unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular. Any example(s) following the term“e.g.” or “for example” is not meant to be exhaustive or limiting.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. In this specification and in the claims that follow,reference will be made to a number of terms that shall be defined tohave the following meanings.

The term “isolated molecule” (where the molecule is, for example, apolypeptide, a polynucleotide, or an antibody or fragment thereof) is amolecule that by virtue of its origin or source of derivation (1) is notassociated with naturally associated components that accompany it in itsnative state, (2) is substantially free of other molecules from the samespecies (3) is expressed by a cell from a different species, or (4) doesnot occur in nature. Thus, a molecule that is chemically synthesized, orexpressed in a cellular system different from the cell from which itnaturally originates, will be “isolated” from its naturally associatedcomponents. A molecule also may be rendered substantially free ofnaturally associated components by isolation, using purificationtechniques well known in the art. Molecule purity or homogeneity may beassayed by a number of means well known in the art. For example, thepurity of a polypeptide sample may be assayed using polyacrylamide gelelectrophoresis and staining of the gel to visualize the polypeptideusing techniques well known in the art. For certain purposes, higherresolution may be provided by using HPLC or other means well known inthe art for purification.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies (e.g., a glycoprotein, including an antibody or receptor)comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.In certain embodiments a substantially pure material is at least 50%pure (i.e., free from contaminants), more preferably, at least 90% pure,more preferably, at least 95% pure, yet more preferably, at least 98%pure, and most preferably, at least 99% pure.

The term “identity,” as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween polypeptide or nucleic acid molecule sequences, as the case maybe, as determined by the match between strings of nucleotide or aminoacid sequences. “Identity” measures the percent of identical matchesbetween two or more sequences with gap alignments addressed by aparticular mathematical model of computer programs (i.e. “algorithms”).

The term “similarity” is a related concept, but in contrast to“identity,” refers to a measure of similarity which includes bothidentical matches and conservative substitution matches. Sinceconservative substitutions apply to polypeptides and not nucleic acidmolecules, similarity only deals with polypeptide sequence comparisons.If two polypeptide sequences have, for example, 10 out of 20 identicalamino acids, and the remainder are all nonconservative substitutions,then the percent identity and similarity would both be 50%. If in thesame example, there are 5 more positions where there are conservativesubstitutions, then the percent identity remains 50%, but the percentsimilarity would be 75% (15 out of 20). Therefore, in cases where thereare conservative substitutions, the degree of similarity between twopolypeptide sequences will be higher than the percent identity betweenthose two sequences.

Polypeptide or antibody “fragments” or “portions” according to theinvention may be made by truncation, e.g. by removal of one or moreamino acids from the N and/or C-terminal ends of a polypeptide. Up to10, up to 20, up to 30, up to 40 or more amino acids may be removed fromthe N and/or C terminal in this way. Fragments may also be generated byone or more internal deletions.

A variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to30 or more amino acid substitutions and/or deletions and/or insertionsfrom the specific sequences and fragments discussed above. “Deletion”variants may comprise the deletion of individual amino acids, deletionof small groups of amino acids such as 2, 3, 4 or 5 amino acids, ordeletion of larger amino acid regions, such as the deletion of specificamino acid domains or other features. “Insertion” variants may comprisethe insertion of individual amino acids, insertion of small groups ofamino acids such as 2, 3, 4 or 5 amino acids, or insertion of largeramino acid regions, such as the insertion of specific amino acid domainsor other features. “Substitution” variants preferably involve thereplacement of one or more amino acids with the same number of aminoacids and making conservative amino acid substitutions. For example, anamino acid may be substituted with an alternative amino acid havingsimilar properties, for example, another basic amino acid, anotheracidic amino acid, another neutral amino acid, another charged aminoacid, another hydrophilic amino acid, another hydrophobic amino acid,another polar amino acid, another aromatic amino acid or anotheraliphatic amino acid.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but framework alterations are alsocontemplated. Conservative substitutions are shown below under theheading of “conservative substitutions.” If such substitutions result ina change in biological activity, then more substantial changes,denominated “exemplary substitutions” shown below, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

Amino Acids and Substitutions

Original Conservative Exemplary Residue Substitutions Substitutionsalanine Ala (A) Val Val; Leu; Ile arginine Arg (R) Lys Lys; Gln; Asnasparagine Asn (N) Gln Gln; His; Asp, Lys; Arg aspartatic Asp (D) GluGlu; Asn cysteine Cys (C) Ser Ser; Ala glutamine Gln (Q) Asn Asn; Gluglutamic Glu (E) Asp Asp; Gln glycine Gly (G) Ala Ala histidine His (H)Arg Asn; Gln; Lys; Arg isoleucine Ile (1) Leu Leu; Val; Met; Ala; Phe;Norleucine leucine Leu (L) Ile Norleucine; lie; Val; Met; Ala; Phelysine Lys (K) Arg Arg; Gln; Asn methionine Met (M) Leu Leu; Phe; Ilephenylalanine Phe (F) Tyr Leu; Val; lie; Ala; Tyr proline Pro (P) AlaAla serine Ser (S) Thr Thr threonine Thr (T) Ser Ser tryptophan Trp (W)Tyr Tyr; Phe tyrosine Tyr (Y) Phe Trp; Phe; Thr; Ser Original ResidueConservative Exemplary Substitutions Substitutions valine Val (V) LeuIle; Leu; Met; Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a beta-sheetor helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   -   i. Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;    -   ii. Polar without charge: Cys, Ser, Thr, Asn, Gln;    -   iii. Acidic (negatively charged): Asp, Glu;    -   iv. Basic (positively charged): Lys, Arg;    -   v. Residues that influence chain orientation: Gly, Pro; and    -   vi. Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

One type of substitution, for example, that may be made is to change oneor more cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. Forexample, there can be a substitution of a non-canonical (e.g., notpreferred or common) cysteine. The substitution can be made in a CDR orframework region of a variable domain or in the constant region of anantibody. In some embodiments, the cysteine is canonical (e.g.,preferred or most common). Any cysteine residue not involved inmaintaining the proper conformation of the antibody also may besubstituted, generally with serine, to improve the oxidative stabilityof the molecule and prevent aberrant cross-linking. Conversely, cysteinebond(s) may be added to the antibody to improve its stability,particularly where the antibody is an antibody fragment such as an Fvfragment.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso, unless otherwise specified, any antigen binding fragment thereofthat competes with the intact antibody for specific binding, fusionproteins comprising an antigen binding fragment, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site. Antigen binding fragments include, for example, Fab,Fab′, F(ab′)2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelidantibodies), fragments including complementarity determining regions(CDRs), single chain variable fragment antibodies (scFv), maxibodies,minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

An antibody includes an antibody of any class, such as IgG, IgA, or IgM(or sub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantregion of its heavy chains (HC), immunoglobulins can be assigned todifferent classes. There are five major classes of immunoglobulins: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂. Theheavy chain constant regions that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The terms “antigen-binding portion” or “antigen-binding fragment” of anantibody (or simply “antibody portion”), as used interchangeably herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen (e.g., αvβ8 integrin). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR), disulfide-linked Fvs (dsFv), andanti-idiotypic (anti-Id) antibodies and intrabodies. Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules (knownas single chain Fv (scFv)); see e.g., Bird et al., Science 242:423-426(1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883(1988)). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding fragment” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993);Poljak et al., 1994, Structure 2:1121-1123).

Antibodies may be derived from any mammal, including, but not limitedto, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., orother animals such as birds (e.g. chickens), fish (e.g., sharks) andcamelids (e.g., llamas).

A “variable region” of an antibody refers to the variable region of theantibody light chain (VL) or the variable region of the antibody heavychain (VH), either alone or in combination. As known in the art, thevariable regions of the heavy and light chains each consist of fourframework regions (FRs) connected by three “complementarity determiningregions (CDRs)” also known as hypervariable regions (HVR) and contributeto the formation of the antigen binding site of antibodies. If variantsof a subject variable region are desired, particularly with substitutionin amino acid residues outside of a CDR region (i.e., in the frameworkregion), appropriate amino acid substitution, preferably, conservativeamino acid substitution, can be identified by comparing the subjectvariable region to the variable regions of other antibodies whichcontain CDR1 and CDR2 sequences in the same canonical class as thesubject variable region (Chothia and Lesk, J. Mol. Biol. 196(4):901-917, 1987).

In certain embodiments, definitive delineation of a CDR andidentification of residues comprising the binding site of an antibody isaccomplished by solving the structure of the antibody and/or solving thestructure of the antibody-ligand complex. In certain embodiments, thatcan be accomplished by any of a variety of techniques known to thoseskilled in the art, such as X-ray crystallography. In certainembodiments, various methods of analysis can be employed to identify orapproximate the CDR regions. In certain embodiments, various methods ofanalysis can be employed to identify or approximate the CDR regions.Examples of such methods include, but are not limited to, the Kabatdefinition, the Chothia definition, the AbM definition, the contactdefinition, and the conformational definition.

There are several numbering methods in the art for numbering the aminoacid residues that form the CDRs. The Kabat numbering method is astandard for numbering the residues in an antibody and is also typicallyused to identify CDRs. See, e.g., Johnson & Wu, 2000, Nucleic AcidsRes., 28: 214-8. The Chothia definition is similar to the Kabatdefinition, but the Chothia definition takes into account positions ofcertain structural loop regions. See, e.g., Chothia et al., 1986, J.Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. TheAbM definition uses an integrated suite of computer programs produced byOxford Molecular Group that model antibody structure. See, e.g., Martinet al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; “AbM™, A ComputerProgram for Modeling Variable Regions of Antibodies,” Oxford, UK; OxfordMolecular, Ltd. The AbM definition models the tertiary structure of anantibody from primary sequence using a combination of knowledgedatabases and ab initio methods, such as those described by Samudrala etal., 1999, “Ab Initio Protein Structure Prediction Using a CombinedHierarchical Approach,” in PROTEINS, Structure, Function and GeneticsSuppl., 3:194-198.

The contact definition is based on an analysis of the available complexcrystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol.,5:732-45. In another approach, referred to herein as the “conformationaldefinition” of CDRs, the positions of the CDRs may be identified as theresidues that make enthalpic contributions to antigen binding. See,e.g., Makabe et al., 2008, Journal of Biological Chemistry,283:1156-1166. Still other CDR boundary definitions may not strictlyfollow one of the above approaches but will nonetheless overlap with atleast a portion of the Kabat CDRs, although they may be shortened orlengthened in light of prediction or experimental findings thatparticular residues or groups of residues do not significantly impactantigen binding. As used herein, a CDR may refer to CDRs defined by anyapproach known in the art, including combinations of approaches. Themethods used herein may utilize CDRs defined according to any of theseapproaches. For any given embodiment containing more than one CDR, theCDRs may be defined in accordance with any of Kabat, Chothia, extended,AbM, contact, and/or conformational definitions.

“Contact residue” as used herein with respect to an antibody or theantigen specifically bound thereby, refers to an amino acid residuepresent on an antibody/antigen comprising at least one heavy atom (i.e.,not hydrogen) that is within 4 Å or less of a heavy atom of an aminoacid residue present on the cognate antibody/antigen.

“Framework” (FR) residues are antibody variable domain residues otherthan the CDR residues. A VH or VL domain framework comprises fourframework sub-regions, FR1, FR2, FR3 and FR4, interspersed with CDRs inthe following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Residues in a variable domain are typically numbered according Kabat,which provides a numbering system used for heavy chain variable domainsor light chain variable domains of the compilation of antibodies. See,Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.Using this numbering system, the actual linear amino acid sequence maycontain fewer or additional amino acids corresponding to a shorteningof, or insertion into, a FR or CDR of the variable domain. For example,a heavy chain variable domain may include a single amino acid insert(residue 52a according to Kabat) after residue 52 of H2 and insertedresidues (e.g. residues 82a, 82b, and 82c, according to Kabat) afterheavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.Various algorithms for assigning Kabat numbering are available. Thealgorithm implemented in the version 2.3.3 release of Abysis(www.abysis.org) can be used to assign Kabat numbering to variableregions CDR-L1, CDR-L2, CDR-L3, CDR-H2, and CDR-H3, and the AbMdefinition can then be used for CDR-H1.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, 1975, Nature 256:495, ormay be made by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,1990, Nature 348:552-554, for example. As used herein, “humanized”antibody refers to forms of non-human (e.g. murine) antibodies that arechimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. Preferably, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. The humanized antibody may compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences, but are included to further refineand optimize antibody performance.

The antibody, or antigen-binding fragment thereof, of the invention maybe affinity matured. For example, an affinity matured antibody can beproduced by procedures known in the art (Marks et al., 1992,Bio/Technology, 10:9-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA91:3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al.,1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol.,154(7):3310-9; Hawkins et al., 1992, J. Mol. Biol., 226:889-896; andWO2004/058184).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen bindingresidues.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody or vice versa. The term also encompasses an antibody comprisinga V region from one individual from one species (e.g., a first mouse)and a constant region from another individual from the same species(e.g., a second mouse).

The term “antigen (Ag)” refers to the molecular entity used forimmunization of an immunocompetent vertebrate to produce the antibody(Ab) that recognizes the Ag or to screen an expression library (e.g.,phage, yeast or ribosome display library, among others). Herein, Ag istermed more broadly and is generally intended to include targetmolecules that are specifically recognized by the Ab, thus includingfragments or mimics of the molecule used in an immunization process forraising the Ab or in library screening for selecting the Ab. Thus, forantibodies of the invention binding to αvβ8 integrin, full-length αvβ8integrin from mammalian species (e.g., human, monkey, mouse, and ratαvβ8 integrin), including monomers and multimers, such as dimers,trimers, etc. thereof, as well as truncated and other variants of αvβ8integrin, are referred to as an antigen.

Generally, the term “epitope” refers to the area or region of an antigen(e.g., a protein, nucleic acid, carbohydrate, or lipid, etc.) to whichan antibody specifically binds, i.e., an area or region in physicalcontact with the antibody. Thus, the term “epitope” refers to thatportion of a molecule capable of being recognized by and bound by anantibody at one or more of the antibody's antigen-binding regions.Typically, an epitope is defined in the context of a molecularinteraction between an “antibody, or antigen-binding portion thereof”(Ab), and its corresponding antigen. Epitopes often consist of a surfacegrouping of molecules such as amino acids or sugar side chains and havespecific three-dimensional structural characteristics as well asspecific charge characteristics. In some embodiments, the epitope can bea protein epitope. Protein epitopes can be linear or conformational. Ina linear epitope, all of the points of interaction between the proteinand the interacting molecule (such as an antibody) occur linearly alongthe primary amino acid sequence of the protein. A “nonlinear epitope” or“conformational epitope” comprises noncontiguous polypeptides (or aminoacids) within the antigenic protein to which an antibody specific to theepitope binds. The term “antigenic epitope” as used herein, is definedas a portion of an antigen to which an antibody can specifically bind asdetermined by any method well known in the art, for example, byconventional immunoassays. Alternatively, during the discovery process,the generation and characterization of antibodies may elucidateinformation about desirable epitopes. From this information, it is thenpossible to competitively screen antibodies for binding to the sameepitope. An approach to achieve this is to conduct competition andcross-competition studies to find antibodies that compete orcross-compete with one another for binding to αvβ8 integrin, e.g., theantibodies compete for binding to the antigen.

An antibody that “preferentially binds” or “specifically binds” (usedinterchangeably herein) to an epitope is a term well understood in theart, and methods to determine such specific or preferential binding arealso well known in the art. A molecule is said to exhibit “specificbinding” or “preferential binding” if it reacts or associates morefrequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell or substance than it does withalternative cells or substances. An antibody “specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. Also, an antibody “specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration to that target in asample than it binds to other substances present in the sample. Forexample, an antibody that specifically or preferentially binds to anαvβ8 integrin epitope is an antibody that binds this epitope withgreater affinity, avidity, more readily, and/or with greater durationthan it binds to other αvβ8 integrin epitopes or non-αvβ8 integrinepitopes. It is also understood by reading this definition, for example,that an antibody (or moiety or epitope) which specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding. “Specific binding” or “preferentialbinding” includes a compound, e.g., a protein, a nucleic acid, anantibody, and the like, which recognizes and binds to a specificmolecule, but does not substantially recognize or bind other moleculesin a sample. For instance, an antibody or a peptide receptor whichrecognizes and binds to a cognate ligand or binding partner (e.g., ananti-αvβ8 integrin antibody that binds αvβ8 integrin) in a sample, butdoes not substantially recognize or bind other molecules in the sample,specifically binds to that cognate ligand or binding partner. Thus,under designated assay conditions, the specified binding moiety (e.g.,an antibody or antigen-binding fragment thereof or a receptor or aligand binding portion thereof) binds preferentially to a particulartarget molecule and does not bind in a significant amount to othercomponents present in a test sample.

A variety of assay formats may be used to select an antibody or peptidethat specifically binds a molecule of interest. For example, solid-phaseELISA immunoassay, immunoprecipitation, Biacore™ (GE Healthcare,Piscataway, N.J.), KinExA, fluorescence-activated cell sorting (FACS),Octet™ (FortéBio, Inc., Menlo Park, Calif.) and Western blot analysisare among many assays that may be used to identify an antibody thatspecifically reacts with an antigen, or antigen-binding fragmentthereof, or a receptor, or ligand binding portion thereof, thatspecifically binds with a cognate ligand or binding partner. Typically,a specific or selective reaction will be at least twice the backgroundsignal or noise, more typically more than 10 times background, even moretypically, more than 50 times background, more typically, more than 100times background, yet more typically, more than 500 times background,even more typically, more than 1000 times background, and even moretypically, more than 10,000 times background. Additionally, an antibodyis said to “specifically bind” an antigen when the equilibriumdissociation constant (K_(D)) is ≤1 preferably ≤100 nM, more preferably≤10 nM, even more preferably, ≤100 pM, yet more preferably, ≤10 pM, andeven more preferably, ≤1 pM. In some embodiments, an antibody is said to“specifically bind” an antigen when the equilibrium dissociationconstant (K_(D)) is ≤7 nM.

The term “binding affinity” is herein used as a measure of the strengthof a noncovalent interaction between two molecules, e.g., and antibody,or fragment thereof, and an antigen. The term “binding affinity” is usedto describe monovalent interactions (intrinsic activity).

Additionally, to determine the binding affinity of anti-αvβ8 integrinantibodies to αvβ8 integrin-expressing cells, cell binding experimentscan be performed to determine the apparent affinity. The apparentaffinity of antibody binding to cells expressing the target can becalculated as the EC₅₀ of equilibrium binding titration curves in whichthe geometric mean fluorescence intensity (gMFI) of the antigen bindingpopulation is quantified by flow cytometry.

Binding affinity between two molecules, e.g. an antibody, or fragmentthereof, and an antigen, through a monovalent interaction may bequantified by determination of the dissociation constant (K_(D)). Inturn, K_(D) can be determined by measurement of the kinetics of complexformation and dissociation using, e.g., the surface plasmon resonance(SPR) method (Biacore). The rate constants corresponding to theassociation and the dissociation of a monovalent complex are referred toas the association rate constants k_(a) (or k_(on)) and dissociationrate constant k_(d) (or k_(off)), respectively. K_(D) is related tok_(a) and k_(d) through the equation K_(D)=k_(d)/k_(a). The value of thedissociation constant can be determined directly by well-known methodsand can be computed even for complex mixtures by methods such as those,for example, set forth in Caceci et al. (1984, Byte 9: 340-362). Forexample, the K_(D) may be established using a double-filternitrocellulose filter binding assay such as that disclosed by Wong &Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standardassays to evaluate the binding ability of ligands such as antibodiestowards target antigens are known in the art, including for example,ELISAs, Western blots, RIAs, and flow cytometry analysis, and otherassays exemplified elsewhere herein. The binding kinetics and bindingaffinity of the antibody also can be assessed by standard assays knownin the art, such as Surface Plasmon Resonance (SPR), e.g. by using aBiacore™ system, or KinExA.

A competitive binding assay can be conducted in which the binding of theantibody to the antigen is compared to the binding of the target byanother ligand of that target, such as another antibody or a solublereceptor that otherwise binds the target. The concentration at which 50%inhibition occurs is known as the K_(i). Under ideal conditions, theK_(i) is equivalent to K_(D). The K_(i) value will never be less thanthe K_(D), so measurement of K_(i) can conveniently be substituted toprovide an upper limit for KID.

Following the above definition, binding affinities associated withdifferent molecular interactions, e.g., comparison of the bindingaffinity of different antibodies for a given antigen, may be compared bycomparison of the K_(D) values for the individual antibody/antigencomplexes. K_(D) values for antibodies or other binding partners can bedetermined using methods well established in the art. One method fordetermining the K_(D) is by using surface plasmon resonance, typicallyusing a biosensor system such as a Biacore® system.

Similarly, the specificity of an interaction may be assessed bydetermination and comparison of the K_(D) value for the interaction ofinterest, e.g., a specific interaction between an antibody and anantigen, with the K_(D) value of an interaction not of interest, e.g., acontrol antibody known not to bind αvβ8 integrin.

An antibody that specifically binds its target may bind its target witha high affinity, that is, exhibiting a low K_(D) as discussed above, andmay bind to other, non-target molecules with a lower affinity. Forexample, the antibody may bind to non-target molecules with a K_(D) of1×10⁻⁶ M or more, more preferably 1×10⁻⁵ M or more, more preferably1×10⁻⁴ M or more, more preferably 1×10⁻³ M or more, even more preferably1×10⁻² M or more. An antibody of the invention is preferably capable ofbinding to its target with an affinity that is at least two-fold,10-fold, 50-fold, 100-fold 200-fold, 500-fold, 1,000-fold or 10,000-foldor greater than its affinity for binding to another non-αvβ8 integrinmolecule.

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or antigen-binding fragment thereof, binds to anepitope in a manner sufficiently similar to the binding of a secondantibody, or antigen-binding fragment thereof, such that the result ofbinding of the first antibody with its cognate epitope is detectablydecreased in the presence of the second antibody compared to the bindingof the first antibody in the absence of the second antibody. Thealternative, where the binding of the second antibody to its epitope isalso detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its cognate epitope or ligand, whether to the same,greater, or lesser extent, the antibodies are said to “cross-compete”with each other for binding of their respective epitope(s). Bothcompeting and cross-competing antibodies are encompassed by the presentinvention. Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof), the skilled artisanwould appreciate, based upon the teachings provided herein, that suchcompeting and/or cross-competing antibodies are encompassed and can beuseful for the methods disclosed herein.

Standard competition assays may be used to determine whether twoantibodies compete with each other. One suitable assay for antibodycompetition involves the use of the Biacore technology, which canmeasure the extent of interactions using surface plasmon resonance (SPR)technology, typically using a biosensor system (such as a BIACORE®system). For example, SPR can be used in an in vitro competitive bindinginhibition assay to determine the ability of one antibody to inhibit thebinding of a second antibody. Another assay for measuring antibodycompetition uses an ELISA-based approach.

Furthermore, a high throughput process for “binning” antibodies basedupon their competition is described in International Patent ApplicationNo. WO2003/48731. Competition is present if one antibody (or fragment)reduces the binding of another antibody (or fragment) to αvβ8 integrin.For example, a sequential binding competition assay may be used, withdifferent antibodies being added sequentially. The first antibody may beadded to reach binding that is close to saturation. Then, the secondantibody is added. If the binding of second antibody to αvβ8 integrin isnot detected, or is significantly reduced (e.g., at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90% reduction) as compared to a parallel assay in theabsence of the first antibody (which value can be set as 100%), the twoantibodies are considered as competing with each other.

In addition, an exemplary antibody epitope binning assay using domainswapping between human and mouse αvβ8 integrin proteins to assesspotential epitopes among several antibodies is provided in Example 9.The skilled artisan would appreciate, armed with the teachings providedherein, that there are a wide variety of assays known in the art thatcan be used to determine the binding to a target of at least twoantibodies relative to each other, and such assays are included herein.

Anti-αvβ8 integrin antibodies may be characterized using methods wellknown in the art. For example, one method is to identify the epitope towhich it binds, or “epitope mapping.” There are many methods known inthe art for mapping and characterizing the location of epitopes onproteins, including solving the crystal structure of an antibody-antigencomplex, competition assays, gene fragment expression assays, andsynthetic peptide-based assays, as described, for example, in Chapter 11of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In anadditional example, epitope mapping can be used to determine thesequence to which an anti-αvβ8 integrin antibody binds. Epitope mappingis commercially available from various sources, for example, PepscanSystems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitopecan be a linear epitope, i.e., contained in a single stretch of aminoacids, or a conformational epitope formed by a three-dimensionalinteraction of amino acids that may not necessarily be contained in asingle stretch. Peptides of varying lengths (e.g., at least 4-6 aminoacids long) can be isolated or synthesized (e.g., recombinantly) andused for binding assays with anti-αvβ8 integrin antibody.

In addition, the epitope to which the anti-αvβ8 integrin antibody bindscan be determined in a systematic screening by using overlappingpeptides derived from the αvβ8 integrin sequence (e.g., a human αvβ8integrin sequence) and determining binding by the antibody. According tothe gene fragment expression assays, the open reading frame encodingαvβ8 integrin can be fragmented either randomly or by specific geneticconstructions and the reactivity of the expressed fragments of αvβ8integrin with the antibody to be tested is determined. The genefragments may, for example, be produced by PCR and then transcribed andtranslated into protein in vitro, in the presence of radioactive aminoacids. The binding of the antibody to the radioactively labeled αvβ8integrin fragments is then determined by immunoprecipitation and gelelectrophoresis.

Certain epitopes can also be identified by using large libraries ofrandom peptide sequences displayed on the surface of phage particles(phage libraries) or yeast (yeast display). Alternatively, a definedlibrary of overlapping peptide fragments can be tested for binding tothe test antibody in simple binding assays. In an additional example,mutagenesis of an antigen, domain swapping experiments and alaninescanning mutagenesis can be performed to identify residues required,sufficient, and/or necessary for epitope binding. For example, alaninescanning mutagenesis experiments can be performed using a mutant αvβ8integrin in which various residues of the αvβ8 integrin polypeptide havebeen replaced with alanine. By assessing binding of the antibody to themutant αvβ8 integrin, the importance of the particular αvβ8 integrinresidues to antibody binding can be assessed.

Yet another method which can be used to characterize an anti-αvβ8integrin antibody is to use competition assays with other antibodiesknown to bind to the same antigen, i.e., various fragments on αvβ8integrin, to determine if an anti-αvβ8 integrin antibody binds to thesame epitope as other antibodies. Competition assays are well known tothose of skill in the art.

Furthermore, the epitope for a given antibody/antigen binding pair canbe defined and characterized at different levels of detail using avariety of experimental and computational epitope mapping methods. Theexperimental methods include mutagenesis, X-ray crystallography, NuclearMagnetic Resonance (NMR) spectroscopy, hydrogen/deuterium exchange MassSpectrometry (H/D-MS) and various competition binding methods well-knownin the art. As each method relies on a unique principle, the descriptionof an epitope is intimately linked to the method by which it has beendetermined. Thus, the epitope for a given antibody/antigen pair will bedefined differently depending on the epitope mapping method employed.

At its most detailed level, the epitope for the interaction between theAg and the Ab can be defined by the spatial coordinates defining theatomic contacts present in the Ag-Ab interaction, as well as informationabout their relative contributions to the binding thermodynamics. At aless detailed level the epitope can be characterized by the spatialcoordinates defining the atomic contacts between the Ag and Ab. At afurther less detailed level the epitope can be characterized by theamino acid residues that it comprises as defined by a specificcriterion, e.g., by distance between atoms (e.g., heavy, i.e.,non-hydrogen atoms) in the Ab and the Ag. At a further less detailedlevel the epitope can be characterized through function, e.g., bycompetition binding with other Abs. The epitope can also be defined moregenerically as comprising amino acid residues for which substitution byanother amino acid will alter the characteristics of the interactionbetween the Ab and Ag (e.g., using alanine scanning).

From the fact that descriptions and definitions of epitopes, dependenton the epitope mapping method used, are obtained at different levels ofdetail, it follows that comparison of epitopes for different Abs on thesame Ag can similarly be conducted at different levels of detail.

Epitopes described at the amino acid level, e.g., determined from anX-ray structure, are said to be identical if they contain the same setof amino acid residues. Epitopes are said to overlap if at least oneamino acid is shared by the epitopes. Epitopes are said to be separate(unique) if no amino acid residue is shared by the epitopes.

Epitopes characterized by competition binding are said to be overlappingif the binding of the corresponding antibodies are mutually exclusive,i.e., binding of one antibody excludes simultaneous or consecutivebinding of the other antibody. The epitopes are said to be separate(unique) if the antigen is able to accommodate binding of bothcorresponding antibodies simultaneously.

The definition of the term “paratope” is derived from the abovedefinition of “epitope” by reversing the perspective. Thus, the term“paratope” refers to the area or region on the antibody whichspecifically binds an antigen, i.e., the amino acid residues on theantibody which make contact with the antigen (αvβ8 integrin, or aportion thereof) as “contact” is defined elsewhere herein.

The epitope and paratope for a given antibody/antigen pair may beidentified by routine methods. For example, the general location of anepitope may be determined by assessing the ability of an antibody tobind to different fragments or variant αvβ8 integrin polypeptides. Thespecific amino acids within αvβ8 integrin that make contact with anantibody (epitope) and the specific amino acids in an antibody that makecontact with αvβ8 integrin (paratope) may also be determined usingroutine methods, such as those described in the examples. For example,the antibody and target molecule may be combined and theantibody/antigen complex may be crystallized. The crystal structure ofthe complex may be determined and used to identify specific sites ofinteraction between the antibody and its target.

An antibody according to the current invention may bind to the sameepitope or domain of αvβ8 integrin (e.g., human αvβ8 integrin) as theantibodies of the invention that are specifically disclosed herein.Analyses and assays that may be used for the purpose of suchidentification include assays assessing the competition for binding ofαvβ8 integrin between the antibody of interest and αvβ8 integrinreceptor, in biological activity assays as described in Examples 1-26.

An antibody, or antigen-binding fragment thereof, may have the abilityto compete or cross-compete with another antibody of the invention forbinding to αvβ8 integrin (e.g., human αvβ8 integrin) as describedherein. For example, an antibody of the invention may compete orcross-compete with antibodies described herein for binding to αvβ8integrin, or to a suitable fragment or variant of αvβ8 integrin that isbound by the antibodies disclosed herein.

That is, if a first antibody competes with a second antibody for bindingto αvβ8 integrin, but it does not compete where the second antibody isfirst bound to αvβ8 integrin, it is deemed to “compete” with the secondantibody (also referred to as unidirectional competition). Where anantibody competes with another antibody regardless of which antibody isfirst bound to αvβ8 integrin, then the antibody “cross-competes” forbinding to αvβ8 integrin with the other antibody. Such competing orcross-competing antibodies can be identified based on their ability tocompete/cross-compete with a known antibody of the invention in standardbinding assays. For example, SPR e.g. by using a Biacore™ system, ELISAassays or flow cytometry may be used to demonstratecompetition/cross-competition. Such competition/cross-competition maysuggest that the two antibodies bind to identical, overlapping orsimilar epitopes.

An antibody of the invention may therefore be identified by a methodthat comprises a binding assay which assesses whether or not a testantibody is able to compete/cross-compete with a reference antibody fora binding site on the target molecule. Methods for carrying outcompetitive binding assays are disclosed herein and/or are well known inthe art. For example, they may involve binding a reference antibody ofthe invention to a target molecule using conditions under which theantibody can bind to the target molecule. The antibody/target complexmay then be exposed to a test/second antibody and the extent to whichthe test antibody is able to displace the reference antibody of theinvention from antibody/target complexes may be assessed. An alternativemethod may involve contacting a test antibody with a target moleculeunder conditions that allow for antibody binding, then adding areference antibody of the invention that is capable of binding thattarget molecule and assessing the extent to which the reference antibodyof the invention is able to displace the test antibody fromantibody/target complexes or to simultaneously bind to the target (i.e.,non-competing antibody).

The ability of a test antibody to inhibit the binding of a referenceantibody of the invention to the target demonstrates that the testantibody can compete with a reference antibody of the invention forbinding to the target and thus that the test antibody binds to the same,or substantially the same, epitope or region on the αvβ8 integrinprotein as the reference antibody of the invention. A test antibody thatis identified as competing with a reference antibody of the invention insuch a method is also an antibody of the present invention. The factthat the test antibody can bind αvβ8 integrin in the same region as areference antibody of the invention and can compete with the referenceantibody of the invention suggests that the test antibody may act as aligand at the same binding site as the antibody of the invention andthat the test antibody may therefore mimic the action of the referenceantibody and is, thus, an antibody of the invention. This can beconfirmed by comparing the activity of αvβ8 integrin in the presence ofthe test antibody with the activity of αvβ8 integrin in the presence ofthe reference antibody under otherwise identical conditions, using anassay as more fully described elsewhere herein.

The reference antibody, or antigen-binding fragment thereof, of theinvention may be an antibody as described herein, e.g., an antibody inTable 1, and any variant, or fragment thereof, as described herein thatretains the ability to bind to αvβ8 integrin.

As stated previously elsewhere herein, specific binding may be assessedwith reference to binding of the antibody to a molecule that is not thetarget. This comparison may be made by comparing the ability of anantibody to bind to the target and to another molecule. This comparisonmay be made as described above in an assessment of K_(D) or K_(i). Theother molecule used in such a comparison may be any molecule that is notthe target molecule. Preferably, the other molecule is not identical tothe target molecule. Preferably the target molecule is not a fragment ofthe target molecule.

The other molecule used to determine specific binding may be unrelatedin structure or function to the target. For example, the other moleculemay be an unrelated material or accompanying material in theenvironment.

The other molecule used to determine specific binding may be anothermolecule involved in the same in vivo pathway as the target molecule,e.g., αvβ8 integrin (e.g., human αvβ8 integrin). By ensuring that theantibody of the invention has specificity for αvβ8 integrin over anothersuch molecule, unwanted in vivo cross-reactivity may be avoided.

The antibody of the invention may retain the ability to bind to somemolecules that are related to the target molecule.

Alternatively, the antibody of the invention may have specificity for aparticular target molecule. For example, it may bind to one targetmolecule as described herein, but may not bind, or may bind withsignificantly reduced affinity to a different target molecule asdescribed herein. For example, a full length mature human αvβ8 integrinmay be used as the target, but the antibody that binds to that targetmay be unable to bind to or may bind with lesser affinity to, e.g. otherαvβ8 integrin proteins from other species, such as other mammalian αvβ8integrin. In some embodiments, the antibody binds to both human andmouse αvβ8 integrin.

An “Fc fusion” protein is a protein wherein one or more polypeptides areoperably linked to an Fc polypeptide. An Fc fusion combines the Fcregion of an immunoglobulin with a fusion partner.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g. from about one to about tenamino acid substitutions, and preferably, from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably, at least about 90% sequence identity therewith, morepreferably, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99% sequence identity therewith.

As known in the art, a “constant region” of an antibody refers to theconstant region of the antibody light chain or the constant region ofthe antibody heavy chain, either alone or in combination.

The terms “IgG Fc region”, “Fc region”, “Fc domain” and “Fc”, asinterchangeably used herein refer to the portion of an IgG molecule thatcorrelates to a crystallizable fragment obtained by papain digestion ofan IgG molecule. As used herein, the terms relate to the constant regionof an antibody excluding the first constant region immunoglobulin domainand further relates to portions of that region. Thus, Fc refers to thelast two constant region immunoglobulin domains of IgA, IgD, and IgG,and the last three constant region immunoglobulin domains of IgE andIgM, and the flexible hinge N-terminal to these domains, or portionsthereof. For IgA and IgM, Fc may include the J chain. For IgG, Fccomprises immunoglobulin domains Cγ2 and Cγ3 (C gamma 2 and C gamma 3)and the hinge between Cγ1 (C gamma 1) and Cy2 (C gamma 2). Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the Eu index ofEdelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 asdescribed in Kabat et al., 1991. Typically, the Fc domain comprises fromabout amino acid residue 236 to about 447 of the human IgG1 constantdomain. An exemplary human wild type IgG1 Fc domain amino acid sequenceis set forth in SEQ ID NO: 81 and SEQ ID NO: 82 (including an optionalterminal lysine (K) residue). Fc polypeptide may refer to this region inisolation, or this region in the context of an antibody, orantigen-binding fragment thereof, or Fc fusion protein.

The heavy chain constant domain comprises the Fc region and furthercomprises the CH1 domain and hinge as well as the CH2 and CH3 (and,optionally, CH4 of IgA and IgE) domains of the IgG heavy chain.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity; phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain orantigen-binding fragment thereof) and can be assessed using variousassays known in the art for evaluating such antibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcγR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (IT AM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain, (see, e.g., Daeron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) and regulation of homeostasis ofimmunoglobulins. Methods of measuring binding to FcRn are known (see,e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie etal., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol.Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

“Effector functions” refer to biological activities attributable to theFc region of an antibody, which vary with the antibody isotype. Examplesof antibody effector functions include: C1q binding and complementdependent cytotoxicity (CDC); Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cellsurface receptors (e.g. B cell receptor); and B cell activation.

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, macrophages, cytotoxic Tcells, and neutrophils. The effector cells may be isolated from a nativesource, e.g., from blood.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. NK cells, neutrophils, andmacrophages) enable these cytotoxic effector cells to bind specificallyto an antigen-bearing target cell and subsequently kill the target cellwith cytotoxins. The primary cells for mediating ADCC, NK cells, expressFcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCCactivity of a molecule of interest, an in vitro ADCC assay, such as thatdescribed in U.S. Pat. No. 5,500,362 or 5,821,337 or 6,737,056 (Presta),may be performed. Useful effector cells for such assays include PBMC andNK cells. Alternatively, or additionally, ADCC activity of the moleculeof interest may be assessed in vivo, e.g., in an animal model such asthat disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656(1998). Additional antibodies with altered Fc region amino acidsequences and increased or decreased ADCC activity are described, e.g.,in U.S. Pat. Nos. 7,923,538, and 7,994,290.

An antibody having an “enhanced ADCC activity” refers to an antibodythat is more effective at mediating ADCC in vitro or in vivo compared tothe parent antibody, wherein the antibody and the parent antibody differin at least one structural aspect, and when the amounts of such antibodyand parent antibody used in the assay are essentially the same. In someembodiments, the antibody and the parent antibody have the same aminoacid sequence, but the antibody is afucosylated while the parentantibody is fucosylated. In some embodiments, ADCC activity will bedetermined using the in vitro ADCC assay as herein disclosed, but otherassays or methods for determining ADCC activity, e.g. in an animal modeletc., are contemplated. In some embodiments, an antibody with enhancedADCC activity has enhanced affinity for Fc gamma RIIIA

An antibody with “altered” FcR binding affinity or ADCC activity is onewhich has either enhanced or diminished FcR binding activity and/or ADCCactivity compared to a parent antibody, wherein the antibody and theparent antibody differ in at least one structural aspect. An antibodythat “displays increased binding” to an FcR binds at least one FcR withbetter affinity than the parent antibody. An antibody that “displaysdecreased binding” to an FcR, binds at least one FcR with lower affinitythan a parent antibody. Such antibodies that display decreased bindingto an FcR may possess little or no appreciable binding to an FcR, e.g.,0-20 percent binding to the FcR compared to a native sequence IgG Fcregion.

“Enhanced affinity for Fc gamma RIIIA” refers to an antibody that hasgreater affinity for Fc gamma RIIIA (also referred to, in someinstances, as CD 16a) than a parent antibody, wherein the antibody andthe parent antibody differ in at least one structural aspect.

“Glycoform” refers to a complex oligosaccharide structure comprisinglinkages of various carbohydrate units. Such structures are describedin, e.g., Essentials of Glycobiology Varki et al., eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1999), which alsoprovides a review of standard glycobiology nomenclature. Such glycoformsinclude, but are not limited to, G2, G1, G0, G-1, and G-2 (see, e.g.,International Patent Publication No. WO 99/22764).

“Glycosylation pattern” is defined as the pattern of carbohydrate unitsthat are covalently attached to a protein (e.g., the glycoform) as wellas to the site(s) to which the glycoform(s) are covalently attached tothe peptide backbone of a protein, more specifically to animmunoglobulin protein.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (Clq) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202: 163 (1996), may be performed. Antibodies withaltered Fc region amino acid sequences and increased or decreased Clqbinding capability are described, e.g., in U.S. Pat. No. 6,194,551 B 1,U.S. Pat. Nos. 7,923,538, 7,994,290 and WO 1999/51642. See also, e.g.,Idusogie et al., J.

As used herein, the terms “wild-type amino acid,” “wild-type IgG,”“wild-type antibody,” or “wild-type mAb,” refer to a sequence of aminoor nucleic acids that occurs naturally within a certain population(e.g., human, mouse, rats, cell, etc.).

The term “αvβ8 integrin,” as used herein, generally refers to a proteincomplex comprising an alpha integrin subunit (e.g., an integrin alpha-Vsubunit, e.g., ITGAV, e.g., comprising the amino acid sequence of SEQ IDNO: 77) and a beta integrin subunit (e.g., an integrin subunit beta 8,e.g., ITGB8, e.g., comprising the amino acid sequence of SEQ ID NO: 78).A “human αvβ8 integrin,” as used herein, generally refers to an αvβ8integrin, e.g., comprising a human ITGAV alpha subunit, e.g., comprisingthe sequence of SEQ ID NO: 77, and a human ITGB8 beta subunit, e.g.,comprising the sequence of SEQ ID NO: 78. A “murine αvβ8 integrin” or“mouse αvβ8 integrin,” as used herein, generally refers to an αvβ8integrin, e.g., comprising a murine ITGAV alpha subunit, e.g.,comprising the sequence of SEQ ID NO: 79, and a murine ITGB8 betasubunit, e.g., comprising the sequence of SEQ ID NO: 80. The term αvβ8integrin typically includes αvβ8 integrin homologs and orthologs,including, but not limited to, human, cynomolgus monkey, rat, rabbit,and mouse. As used herein, “αvβ8 integrin” typically refers to amammalian αvβ8 integrin, e.g., human, rat, mouse, non-human primate,bovine, ovine, or porcine αvβ8 integrin (e.g., comprising an integrinalpha-V subunit and an integrin beta 8 subunit from human, rat, mouse,non-human primate, bovine, ovine, or porcine, respectively).Non-limiting exemplary examples of integrin alpha-V subunits includehuman (see, e.g., Genbank Accession Number P06756.2, SEQ ID NO: 77),cynomolgus monkey (see, e.g., SEQ ID NO:84), and mouse (see, e.g., SEQID NO: 79) αvβ8 integrin. Non-limiting exemplary examples of integrinbeta 8 subunits include human (see, e.g., Genbank Accession NumberP26012.1, SEQ ID NO: 78), cynomolgus monkey (see, e.g., SEQ ID NO: 85),and mouse (see, e.g., SEQ ID NO:80) αvβ8 integrin. The term “αvβ8integrin” also encompasses fragments, variants, isoforms, and otherhomologs of such αvβ8 integrin subunit molecules. Variant αvβ8 integrinmolecules will generally be characterized by having the same type ofactivity as naturally occurring αvβ8 integrin, such as the ability tobind an αvβ8 integrin ligand, e.g., as described herein, the ability toinduce receptor-mediated activity, and the ability to bind, or not, theantibody, or antigen-fragment thereof, of the invention.

Exemplary amino acid and nucleotide sequences for TGFβ and LAP are knownin the art. For example, a precursor polypeptide comprising TGFβ1 andLAP (e.g., human sequence UniProt Accession No. P01137) ispost-translationally processed into about amino acids 30-278 of UniProtAccession No. P01137 corresponding to LAP and about amino acids 279-390of UniProt Accession No. P01137 corresponding to human TGFβ1. Similarly,a precursor polypeptide comprising TGFβ3 and LAP (e.g., human sequenceUniProt Accession No. P10600) is post-translationally processed intoabout amino acids 24-300 of UniProt Accession No. P10600 correspondingto LAP and about amino acids 301-412 of UniProt Accession No. 10600corresponding to human TGFβ3.

The αvβ8 integrin may comprise one or more, two or more, three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more, ten or more, twelve or more or fifteen or more surfaceaccessible residues of αvβ8 integrin. The target molecule may comprise aknown epitope from αvβ8 integrin.

As outlined elsewhere herein, certain positions of the antibody moleculecan be altered. By “position” as used herein is meant a location in thesequence of a protein. Positions may be numbered sequentially, oraccording to an established format, for example the EU index and Kabatindex can be used to number amino acid residues of an antibody. Forexample, position 297 is a position in the human antibody IgG1.Corresponding positions are determined as outlined above, generallythrough alignment with other parent sequences.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297, also referred to as N297) is a residue in thehuman antibody IgG1.

As known in the art, “polynucleotide,” or “nucleic acid,” as usedinterchangeably herein, refer to chains of nucleotides of any length,and include DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a chain by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thechain. The sequence of nucleotides may be interrupted by non-nucleotidecomponents. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications include, for example, “caps”, substitution of oneor more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping group moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. One or more phosphodiesterlinkages may be replaced by alternative linking groups. Thesealternative linking groups include, but are not limited to, embodimentswherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”),(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in whicheach R or R′ is independently H or substituted or unsubstituted alkyl(1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl,cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. The preceding description applies toall polynucleotides referred to herein, including RNA and DNA.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected and/or transformed in vivo with a polynucleotide of thisinvention.

Host cells may be prokaryotic cells or eukaryotic cells. Exemplaryeukaryotic cells include mammalian cells, such as primate or non-primateanimal cells; fungal cells, such as yeast; plant cells; and insectcells.

Any host cell susceptible to cell culture, and to expression of proteinor polypeptides, may be utilized in accordance with the presentinvention. In certain embodiments, the host cell is mammalian. Mammaliancell lines available as hosts for expression are well known in the artand include many immortalized cell lines available from the AmericanType Culture Collection (ATCC). Nonlimiting exemplary mammalian cellsinclude, but are not limited to, NS0 cells, HEK 293 and Chinese hamsterovary (CHO) cells, and their derivatives, such as 293-6E and CHO DG44cells, CHO DXB11, and Potelligent® CHOK1SV cells (BioWa/Lonza,Allendale, N.J.). Mammalian host cells also include, but are not limitedto, human cervical carcinoma cells (HeLa, ATCC CCL 2), baby hamsterkidney (BHK, ATCC CCL 10) cells, monkey kidney cells (COS), and humanhepatocellular carcinoma cells (e.g., Hep G2). Other non-limitingexamples of mammalian cells that may be used in accordance with thepresent invention include human retinoblasts (PER.C6®; CruCell, Leiden,The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7,ATCC CRL 1651); human embryonic kidney line 293 (HEK 293) or 293 cellssubcloned for growth in suspension culture (Graham et al., 1977, J. GenVirol. 36:59); mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod.23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1 587); canine kidney cells (MDCK, ATCCCCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lungcells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mousemammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., 1982,Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; a humanhepatoma line (Hep G2); and numerous myeloma cell lines, including, butnot limited to, BALB/c mouse myeloma line (NS0/1, ECACC No: 85110503),NS0 cells and Sp2/0 cells.

Additionally, any number of commercially and non-commercially availablecell lines that express polypeptides or proteins may be utilized inaccordance with the present invention. One skilled in the art willappreciate that different cell lines might have different nutritionrequirements and/or might require different culture conditions foroptimal growth and polypeptide or protein expression, and will be ableto modify conditions as needed.

The invention includes any eukaryotic expression system known in the artor disclosed herein for production of proteins of interest, such asexpression in an insect cell system, a yeast expression system, or amammalian cell system, such as, but not limited to, CHO cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

By the term “leader peptide” or “leader sequence” or “leader signalsequence” or “signal sequence”, as used interchangeably herein, is meantany nucleic acid sequence, or amino acid sequence encoded thereby, thatmay be present on the 5′ end of a nucleic acid molecule and/or at ornear the N-terminus of a polypeptide, that when present may mediate thetransport of the polypeptide to an organelle of destination, including,but not limited to, the secretion of the polypeptide from a cell. Suchleader sequences include, but are not limited to, nucleic acid sequencescomprising, e.g., ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGCGTGCACTCC (SEQ ID NO: 187), and amino acid sequences encoded thereby, suchas, but not limited to, MGWSCIILFLVATATGVHS (SEQ ID NO: 188). Theinvention encompasses these and any other leader signals (nucleic andamino acid sequences) known in the art or to be identified which canresult in the transport of a polypeptide to the desired organelle, e.g.,the endoplasmic reticulum, and/or secreted from the cell. Generally, thesignal peptide is removed from and/or is not present in the maturepolypeptide.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: improved survival rate (reduced mortality), reduction ininflammatory response to the disease, reduction in the amount of tissuefibrosis, improvement in the appearance of the disease lesions,limitation of the pathological lesions to focal sites, decreased extentof damage from the disease, decreased duration of the disease, and/orreduction in the number, extent, or duration of symptoms related to thedisease. The term includes the administration of the compounds or agentsof the present invention to prevent or delay the onset of the symptoms,complications, or biochemical indicia of a disease, alleviating thesymptoms or arresting or inhibiting further development of the disease,condition, or disorder. Treatment may be prophylactic (to prevent ordelay the onset of the disease, or to prevent the manifestation ofclinical or subclinical symptoms thereof) or therapeutic suppression oralleviation of symptoms after the manifestation of the disease. In someembodiments, the disease, condition or disorder is a cancer.

As used herein, the term “cancer” is meant to include all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. Examples of cancerousdisorders include, but are not limited to, solid tumors, hematologicalcancers, soft tissue tumors, and metastatic lesions. Examples of solidtumors include malignancies, e.g., sarcomas, and carcinomas (includingadenocarcinomas and squamous cell carcinomas), of the various organsystems, such as those affecting liver, lung, breast, lymphoid,gastrointestinal (e.g., colon), genitourinary tract (e.g., renal,urothelial cells), prostate and pharynx. Adenocarcinomas includemalignancies such as most colon cancers, rectal cancer, renal-cellcarcinoma, liver cancer, non-small cell carcinoma of the lung, cancer ofthe small intestine and cancer of the esophagus. Squamous cellcarcinomas include malignancies, e.g., in the lung, esophagus, skin,head and neck region, oral cavity, anus, and cervix. In one embodiment,the cancer is a melanoma, e.g., an advanced stage melanoma. Metastaticlesions of the aforementioned cancers can also be treated using themethods and compositions of the invention. Exemplary cancers whosegrowth can be treated, e.g., reduced, using the antibodies moleculesdisclosed herein include cancers typically responsive to immunotherapy.

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering an anti-αvβ8 integrin antibody.“Ameliorating” also includes shortening or reduction in duration of asymptom.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toaffect any one or more beneficial or desired results. In more specificaspects, an effective amount prevents, alleviates or amelioratessymptoms of disease, e.g., a cancer, and/or prolongs the survival of thesubject being treated. For prophylactic use, beneficial or desiredresults include eliminating or reducing the risk, lessening theseverity, or delaying the outset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as reducing one or more symptomsof a αvβ8 integrin-mediated disease, disorder or condition, decreasingthe dose of other medications required to treat the disease, enhancingthe effect of another medication, and/or delaying the progression of thedisease of patients. An effective dosage can be administered in one ormore administrations. For purposes of this invention, an effectivedosage of drug, compound, or pharmaceutical composition is an amountsufficient to accomplish prophylactic or therapeutic treatment eitherdirectly or indirectly. As is understood in the clinical context, aneffective dosage of a drug, compound, or pharmaceutical composition mayor may not be achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective dosage” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

The antibodies, or antigen-binding fragments thereof, can beadministered in combination with one or more therapies (e.g., referredto herein as a “second therapy”). By “in combination with,” it is notintended to imply that the therapy or the therapeutic agents must beadministered at the same time and/or formulated for delivery together,although these methods of delivery are within the scope describedherein. The anti-αvβ8 integrin antibodies, or antigen-binding fragmentsthereof, can be administered concurrently with, prior to, or subsequentto, one or more other additional therapies or therapeutic agents. Theanti-αvβ8 integrin antibodies, or antigen-binding fragments thereof, andthe second therapy, e.g., other agent or therapeutic protocol, can beadministered in any order. In general, each agent will be administeredat a dose and/or on a time schedule determined for that agent. In willfurther be appreciated that the additional therapeutic agent utilized inthis combination may be administered together in a single composition oradministered separately in different compositions. In some embodiments,the levels utilized in combination will be lower than those utilizedindividually.

A “synergistic combination” or a combination that acts“synergistically,” is a combination that exhibits increased effects thatare not predicted when compared with a merely additive effect of theindividual therapies combined.

An “individual” or a “subject” is a mammal, more preferably, a human.Mammals also include, but are not limited to, farm animals (e.g., cows,pigs, horses, chickens, etc.), sport animals, pets, primates, horses,dogs, cats, mice and rats. In some embodiments, the individual is atrisk for a disease, disorder or condition mediated by or associated withαvβ8 integrin binding to its receptor and signaling mediated thereby. Incertain embodiments, the subject has a disorder or condition asdescribed herein, e.g., a cancer.

As used herein, “pharmaceutically acceptable carrier” or “pharmaceuticalacceptable excipient” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline (PBS) or normal (0.9%) saline. Compositions comprising suchcarriers are formulated by known conventional methods (see, for example,Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., MackPublishing Co., Easton, Pa., 1990; and Remington, The Science andPractice of Pharmacy 20th Ed. Mack Publishing, 2000).

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

II. ANTI-αVβ8 INTEGRIN ANTIBODIES

The present invention relates to antibodies and antigen-bindingfragments thereof, that bind to αvβ8 integrin. Preferably, theantibodies specifically bind to αvβ8 integrin, i.e., they bind to αvβ8integrin but they do not detectably bind, or bind at a lower affinity,to other αv integrins (e.g., αvβ3 integrin, αvβ5 integrin and αvβ6integrin). The invention further relates to anti-αvβ8 integrinantibodies that exhibit an altered effector function. In someembodiments, the altered effector function is decreased ADCC. In someembodiments, the altered effector function is decreased CDC. Theinvention also relates to compositions comprising such antibodies aswell as uses for such antibodies, including therapeutic andpharmaceutical uses.

In one embodiment, the disclosure provides any of the following, orcompositions (including pharmaceutical compositions) comprising, anantibody having a light chain sequence, or a fragment thereof, and aheavy chain, or a fragment thereof, derived from, but not identical to,the mouse hybridoma antibody ADWA-11 (also referred to as ADWA11,mADWA11, mADWA-11), as disclosed in U.S. Pat. No. 9,969,804, which isherein incorporated by reference in its entirety, and as set forth in,e.g., SEQ ID NO: 20-33 and 71-76 of the present description.

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody fragment (e.g., a domain antibody),humanized antibodies, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. The antibodies may be murine, rat, human, or anyother origin (including chimeric or humanized antibodies). In someembodiments, the anti-αvβ8 integrin antibody is a monoclonal antibody.In some embodiments, the anti-αvβ8 integrin antibody is a human orhumanized antibody. In some embodiments, the anti-αvβ8 integrin antibodyis a chimeric antibody.

The anti-αvβ8 integrin antibodies of the invention may be made by anymethod known in the art. General techniques for production of human andmouse antibodies are known in the art and/or are described herein.

Following initial identification, the activity of a candidate anti-αvβ8integrin antibody can be further confirmed and refined by bioassays,known to test the targeted biological activities. In some embodiments,an in vitro cell assay is used to further characterize a candidateanti-αvβ8 integrin antibody. For example, bioassays can be used toscreen candidates directly. Some of the methods for identifying andcharacterizing an anti-αvβ8 integrin antibody are described in detail inthe Examples.

Table 1 below is a summary of amino acid and nucleotide sequences forthe murine, chimeric, and humanized anti-αvβ8 integrin antibodies, e.g.,as described herein. The amino acid and nucleotide sequences of theheavy and light chain CDRs, the amino acid and nucleotide sequences ofthe heavy and light chain variable regions, and the amino acid andnucleotide sequences of the heavy and light chains are shown in thisTable. Generally, unless specifically indicated, the anti-αvβ8 integrinantibodies of the invention can include any combination of one or moreKabat CDRs and/or Chothia hypervariable loops as set forth in Table 1.In some embodiments, the anti-αvβ8 integrin antibodies of the inventioncan include any combination of one or more VH and/or VL sequences as setforth in Table 1. In some embodiments, the anti-αvβ8 integrin antibodiesof the invention can include any combination of one or more frameworkregions (e.g., FR1, FR2, FR3, and FR4) as described in Table 1. It maybe generally understood, and as indicated in Table 1, each VH and VLsequence typically includes three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

In some embodiments, where an anti-αvβ8 integrin antibody comprises aC-terminal lysine (K) amino acid residue on a heavy chain polypeptide(e.g., human IgG1 heavy chain comprises a terminal lysine), one skilledin the art would understand that the lysine residue may be clippedresulting in an antibody with a heavy chain lacking the C-terminallysine residue. Additionally, the antibody heavy chain may be producedusing a nucleic acid that does not encode the lysine. Thus, in someembodiments, an anti-αvβ8 integrin antibody comprises a heavy chainwhere the terminal lysine otherwise present is not present.

TABLE 1Amino acid and nucleotide sequences for αvβ8 integrin antibodies andother peptides. SEQ ID Name NO. Sequence ADWA11 2.4 VL   7DIQMTQSPSSLSASVGDRVTITCRSTKSLSHFNGNTYL VL amino acidFWYQQKPGKAPKRLIYYMSSLASGVPSRFSGSGSGTDF sequenceTLTISSLQPEDFATYYCQQSLEYPFTFGGGTKVEIK The underlined aminoacid residues are the CDR sequences according to Kabat(also referred to as ADWA11_VK01_2.4) ADWA11 2.4 CDR-L1  11RSTKSLSHFNGNTYLF according to Kabat ADWA11 2.4 CDR-L2  12 YYMSSLASaccording to Kabat ADWA11 2.4 CDR-L3  13 QQSLEYPFT according to KabatADWA11 2.4 CDR-L1  17 STKSLSHFNGNTYL according to ChothiaADWA11 2.4 CDR-L2  18 YYMSS according to Chothia ADWA11 2.4 CDR-L3  19QSLEYPFT according to Chothia ADWA11 2.4 VH   6EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVR VH amino acidQAPGKGLEWVGWIDPDQGNTIYEPKFQGRFTISADTSK sequenceNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS The underlined amino Sacid residues are the CDR sequences according to Kabat ADWA11 2.4 CDR-H1  8 DYYMN according to Kabat ADWA11 2.4 CDR-H2   9 WIDPDQGNTIYEPKFQGaccording to Kabat ADWA11 2.4 CDR-H3  10 RLLMDY according to KabatADWA11 2.4 CDR-H1  14 GFNIKDYYMN according to Chothia ADWA11 2.4 CDR-H2 15 WIDPDQGN according to Chothia ADWA11 2.4 CDR-H3  16 RLLMDYaccording to Chothia ADWA11 2.4   5DIQMTQSPSSLSASVGDRVTITCRSTKSLSHFNGNTYL Light chain (LC)FWYQQKPGKAPKRLIYYMSSLASGVPSRFSGSGSGTDF amino acid sequenceTLTISSLQPEDFATYYCQQSLEYPFTFGGGTKVEIKRT VL sequence isVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ underlinedWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGECADWA11 2.4 Heavy   2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRchain (HC) amino QAPGKGLEWVGWIDPDQGNTIYEPKFQGRFTISADTSK acid sequenceNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS VH sequence isSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV underlinedTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKADWA11 2.4 Heavy   3 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRchain amino acid QAPGKGLEWVGWIDPDQGNTIYEPKFQGRFTISADTSK sequence withoutNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS terminal lysineSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV residueTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS VH sequence isLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP underlinedAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGADWA11 2.4 Light   4 atgggatggagctgtatcatcctcttcttggtagcaacchain DNA sequence agctacaggcgtgcactccGACATCCAGATGACCCAGT Nucleic acidCCCCTTCCAGCCTGAGCGCTTCCGTGGGCGACAGGGTG residues encodingACCATCACCTGCAGGTCCACCAAGTCCCTGTCCCACTT the VL areCAACGGCAACACCTACCTGTTCTGGTACCAGCAGAAGC underlinedCCGGCAAGGCCCCCAAGAGGCTGATCTACTACATGTCC Nucleic acidTCCCTGGCCTCCGGAGTGCCCTCCAGGTTCTCCGGATC residues encodingCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCC the leader are inTGCAGCCCGAGGATTTCGCCACCTACTACTGCCAGCAG lowercase lettersTCCCTGGAGTACCCCTTCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC GTCACAAAGAGCTTCAACAGGGGAGAGTGTADWA11 2.4 Light 185 GACATCCAGATGACCCAGTCCCCTTCCAGCCTGAGCGCchain DNA sequence TTCCGTGGGCGACAGGGTGACCATCACCTGCAGGTCCA Nucleic acidCCAAGTCCCTGTCCCACTTCAACGGCAACACCTACCTG residues encodingTTCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGAG the VL areGCTGATCTACTACATGTCCTCCCTGGCCTCCGGAGTGC underlinedCCTCCAGGTTCTCCGGATCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGATTTCGCCACCTACTACTGCCAGCAGTCCCTGGAGTACCCCTTCACCTTCGGCGGCGGCACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGT ADWA11 2.4 186GACATCCAGATGACCCAGTCCCCTTCCAGCCTGAGCGC VL DNA sequenceTTCCGTGGGCGACAGGGTGACCATCACCTGCAGGTCCACCAAGTCCCTGTCCCACTTCAACGGCAACACCTACCTGTTCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGAGGCTGATCTACTACATGTCCTCCCTGGCCTCCGGAGTGCCCTCCAGGTTCTCCGGATCCGGCTCCGGCACCGACTTCACCCTGACCATCTCCTCCCTGCAGCCCGAGGATTTCGCCACCTACTACTGCCAGCAGTCCCTGGAGTACCCCTTCA CCTTCGGCGGCGGCACCAAGGTGGAGATCAAAADWA11 2.4 Light 187 ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACchain and heavy AGCTACAGGCGTGCACTCC chain leader DNA sequenceADWA11 2.4 Light 188 MGWSCIILFLVATATGVHS chain and heavychain leader amino acid sequence ADWA11 2.4   1atgggatggagctgtatcatcctcttcttggtagcaac Heavy Chain DNAagctacaggcgtgcactccGAGGTGCAGCTGGTGGAAA sequence (withGCGGAGGAGGCCTGGTGCAGCCTGGAGGAAGCCTGAGG terminal lysine)CTGAGCTGTGCCGCCAGCGGCTTCAACATCAAGGACTA Nucleic acidCTACATGAACTGGGTGAGGCAGGCCCCTGGCAAAGGAC residues encodingTGGAGTGGGTGGGCTGGATCGACCCCGACCAGGGCAAC the VH areACCATCTACGAGCCCAAGTTCCAGGGCAGGTTCACCAT underlinedCAGCGCCGACACCAGCAAGAACAGCGCCTACCTGCAGA Nucleic acidTGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTAC residues encodingTGCGCCAGGAGGCTGCTGATGGACTACTGGGGCCAGGG the leader are inCACACTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCC lower case lettersCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCCCCCGGAAAAADWA11 2.4 189 GAGGTGCAGCTGGTGGAAAGCGGAGGAGGCCTGGTGCA Heavy Chain DNAGCCTGGAGGAAGCCTGAGGCTGAGCTGTGCCGCCAGCG sequence (withGCTTCAACATCAAGGACTACTACATGAACTGGGTGAGG terminal lysine)CAGGCCCCTGGCAAAGGACTGGAGTGGGTGGGCTGGAT Nucleic acidCGACCCCGACCAGGGCAACACCATCTACGAGCCCAAGT residues encodingTCCAGGGCAGGTTCACCATCAGCGCCGACACCAGCAAG the VH areAACAGCGCCTACCTGCAGATGAACTCCCTGAGGGCCGA underlinedGGACACCGCCGTGTACTACTGCGCCAGGAGGCTGCTGATGGACTACTGGGGCCAGGGCACACTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCG GAAAA ADWA11 2.4 190GAGGTGCAGCTGGTGGAAAGCGGAGGAGGCCTGGTGCA VH DNA sequenceGCCTGGAGGAAGCCTGAGGCTGAGCTGTGCCGCCAGCGGCTTCAACATCAAGGACTACTACATGAACTGGGTGAGGCAGGCCCCTGGCAAAGGACTGGAGTGGGTGGGCTGGATCGACCCCGACCAGGGCAACACCATCTACGAGCCCAAGTTCCAGGGCAGGTTCACCATCAGCGCCGACACCAGCAAGAACAGCGCCTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCTGCTGATGGACTACTGGGGCCAGGGCACACTGGTCACCGTCTCC TCA ADWA11 2.4 183atgggatggagctgtatcatcctcttcttggtagcaac Heavy Chain DNAagctacaggcgtgcactccGAGGTGCAGCTGGTGGAAA sequenceGCGGAGGAGGCCTGGTGCAGCCTGGAGGAAGCCTGAGG Nucleic acid withoutCTGAGCTGTGCCGCCAGCGGCTTCAACATCAAGGACTA terminal lysine,CTACATGAACTGGGTGAGGCAGGCCCCTGGCAAAGGAC residues encodingTGGAGTGGGTGGGCTGGATCGACCCCGACCAGGGCAAC the VH areACCATCTACGAGCCCAAGTTCCAGGGCAGGTTCACCAT underlinedCAGCGCCGACACCAGCAAGAACAGCGCCTACCTGCAGA Nucleic acidTGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTAC residues encodingTGCGCCAGGAGGCTGCTGATGGACTACTGGGGCCAGGG the leader are inCACACTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCC lower caseCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCCCCCGGA ADWA11 2.4191 GAGGTGCAGCTGGTGGAAAGCGGAGGAGGCCTGGTGCA Heavy Chain DNAGCCTGGAGGAAGCCTGAGGCTGAGCTGTGCCGCCAGCG sequenceGCTTCAACATCAAGGACTACTACATGAACTGGGTGAGG Nucleic acid withoutCAGGCCCCTGGCAAAGGACTGGAGTGGGTGGGCTGGAT terminal lysine,CGACCCCGACCAGGGCAACACCATCTACGAGCCCAAGT residues encodingTCCAGGGCAGGTTCACCATCAGCGCCGACACCAGCAAG the VH areAACAGCGCCTACCTGCAGATGAACTCCCTGAGGGCCGA underlinedGGACACCGCCGTGTACTACTGCGCCAGGAGGCTGCTGATGGACTACTGGGGCCAGGGCACACTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCG GA Mouse hybridoma  21DIVMTQAAPSVPVTPGESVSISCRSTKSLLHFNGNTYL antibody ADWA-11FWFLQRPGQSPQRLIYYMSNLASGVPDRFSGRGSGTDF VL amino acidTLRISRVEAEDVGVYYCMQSLEYPFTFGTGTKLEIK sequence The underlined aminoacid residues are the CDR sequences according to Kabat Mouse hybridoma 20 EVQLQQSGAELVRPGAFVKLSCKASGFNIKDYYMNWVL antibody ADWA-11QRPEQGLEWIGWIDPDNGNTIYDPKFQGKASITADTSS VH amino acidNTAYLQLSSLTSEDTAVYYCARRLLMDYWGQGTSVTVS sequence S The underlined aminoacid residues are the CDR sequences according to Kabat Mouse hybridoma 25 RSTKSLLHFNGNTYLF antibody ADWA-11 CDR-L1 according to KabatMouse hybridoma  26 YYMSNLAS antibody ADWA-11 CDR-L2 according to KabatMouse hybridoma  27 MQSLEYPFT antibody ADWA-11 CDR-L3 according to KabatMouse hybridoma  71 RSTKSLLHFNGNTYLF antibody ADWA-11 AlternateAlternate CDR-L1 according to Kabat Mouse hybridoma  72 YYMSNLASantibody ADWA-11 Alternate Alternate CDR-L2 according to KabatMouse hybridoma  73 MQSLEYPFT antibody ADWA-11 AlternateAlternate CDR-L3 according to Kabat Mouse hybridoma  22 DYYMNantibody ADWA-11 CDR-H1 according to Kabat Mouse hybridoma  23WIDPDNGNTIYDPKFQG antibody ADWA-11 CDR-H2 according to KabatMouse hybridoma  24 RLLMDY antibody ADWA-11 CDR-H3 according to KabatMouse hybridoma  31 STKSLLHFNGNTYL antibody ADWA-11 CDR-L1 according toChothia Mouse hybridoma  32 YYMSN antibody ADWA-11 CDR-L2 according toChothia Mouse hybridoma  33 QSLEYPFT antibody ADWA-11CDR-L3 according to Chothia Mouse hybridoma  74 STKSLLHFNGNTYLantibody ADWA-11 Alternate Alternate CDR-L1 according to ChothiaMouse hybridoma  75 YYMSN antibody ADWA-11 Alternate Alternate CDR-L2according to Chothia Mouse hybridoma  76 QSLEYPFT antibody ADWA-11Alternate Alternate CDR-L3 according to Chothia Mouse hybridoma  28GFNIKDYYMN antibody ADWA-11 CDR-H1 according to Chothia Mouse hybridoma 29 WIDPDNGN antibody ADWA-11 CDR-H2 according to ChothiaMouse hybridoma  30 RLLMDY antibody ADWA-11 CDR-H3 according to ChothiaADWA11_VK01 (1)  47 DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL(also referred to FWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF herein asTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK adwa_VL_1.1 L46R) VL amino acidsequence ADWA11_VK01_1a (1)  48 DIQMTQSPSSLSASVGDRVTITCRSTKSILHFNGNTYLL29I FWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK sequence ADWA11_VK01_1b (1)  49DIQMTQSPSSLSASVGDRVTITCRSTKSLSHFNGNTYL L30SFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK sequence ADWA11_VK01_1c (1)  50DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNSYL T36SFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK sequence ADWA11_VK01_2a (1)  51DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL Y55AFWYQQKPGKAPKRLIYAMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK sequence ADWA11_VK01_2b (1)  52DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL M56AFWYQQKPGKAPKRLIYYASNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK sequence ADWA11_VK01_2c (1)  53DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL N58SFWYQQKPGKAPKRLIYYMSSLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK sequence ADWA11_VK01_2d (1)  54DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL A60QFWYQQKPGKAPKRLIYYMSNLQSGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_3a (1)  55DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL M94QFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCQQSLEYPFTFGQGTKVEIK sequence ADWA11_VK01_3b (1)  56DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL L97YFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSYEYPFTFGQGTKVEIK sequence ADWA11_VK01_3c (1)  57DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL E98SFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLSYPFTFGQGTKVEIK sequence ADWA11_VK01_3d (1)  58DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL Y99TFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLETPFTFGQGTKVEIK sequence ADWA11_VK01_4a (1)  59DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL F101LFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLEYPLTFGQGTKVEIK sequence ADWA11_VK01_4b (1)  60DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL F101WFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLEYPWTFGQGTKVEIK sequence ADWA11_VK01_4c (1)  61DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL Q105GFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF VL amino acidTLTISSLQPEDFATYYCMQSLEYPFTFGGGTKVEIK sequence ADWA11VK1 IGKV2-28  62DIVMTQSPLSLPVTPGEPASISCRSTKSLLHFNGNTYL VL amino acidFWYLQKPGQSPQLLIYYMSNLASGVPDRFSGSGSGTDF sequenceTLKISRVEAEDVGVYYCMQSLEYPFTFGQGTKVEIK ADWA11VK2 IGKV2-30  63DVVMTQSPLSLPVTLGQPASISCRSTKSLLHFNGNTYL VL amino acidFWFQQRPGQSPRRLIYYMSNLASGVPDRFSGSGSGTDF sequenceTLKISRVEAEDVGVYYCMQSLEYPFTFGQGTKVEIK ADWA11VK3 IGKV4-1  64DIVMTQSPDSLAVSLGERATINCRSTKSLLHFNGNTYL VL amino acidFWYQQKPGQPPKLLIYYMSNLASGVPDRFSGSGSGTDF sequenceTLTISSLQAEDVAVYYCMQSLEYPFTFGQGTKVEIK ADWA11VK4 IGKV1-39  65DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL VL amino acidFWYQQKPGKAPKLLIYYMSNLASGVPSRFSGSGSGTDF sequenceTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK ADWA11VK5 IGKV3-11  66EIVLTQSPATLSLSPGERATLSCRSTKSLLHFNGNTYL VL amino acidFWYQQKPGQAPRLLIYYMSNLASGIPARFSGSGSGTDF sequenceTLTISSLEPEDFAVYYCMQSLEYPFTFGQGTKVEIK ADWA11_VK01_2.1  67DIQMTQSPSSLSASVGDRVTITCRSTKSLSHFNGNTYL VL amino acidFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF sequenceTLTISSLQPEDFATYYCQQSLEYPFTFGGGTKVEIK ADWA11_VK01_2.2  68DIQMTQSPSSLSASVGDRVTITCRSTKSLSHFNGNTYL VL amino acidFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDF sequenceTLTISSLQPEDFATYYCMQSYEYPFTFGGGTKVEIK ADWA11_VK01_2.3  69DIQMTQSPSSLSASVGDRVTITCRSTKSLSHFNGNTYL VL amino acidFWYQQKPGKAPKRLIYYASNLASGVPSRFSGSGSGTDF sequenceTLTISSLQPEDFATYYCQQSLEYPFTFGGGTKVEIK ADWA11VH1 IGHV1-46  34QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMNWVR VH amino acidQAPGQGLEWIGWIDPDNGNTIYDQKFQGRVTMTRDTST sequenceSTVYMELSSLRSEDTAVYYCARRLLMDYWGQGTLVTVS S ADWA11VH2 IGHV3-23  35EVQLLESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVR VH amino acidQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTISRDNSK sequenceNTLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S ADWA11VH3 IGHV3-30  36QVQLVESGGGVVQPGRSLRLSCAASGFNIKDYYMNWVR VH amino acidQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTISRDNSK sequenceNTLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S ADWA11VH4 IGHV1-69  37QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDYYMNWVR VH amino acidQAPGQGLEWIGWIDPDNGNTIYDQKFQGRVTITADEST sequenceSTAYMELSSLRSEDTAVYYCARRLLMDYWGQGTLVTVS ADWA11VH5 IGHV3-48  38EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVR VH amino acidQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTISRDNAK sequenceNSLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S ADWA11 VH05_VK1  39EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVR (also referred toQAPGKGLEWVGWIDPDNGNTIYDPKFQGRFTISADTSK herein asNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS adwa_VH_1.5 T28N + SF29I + R72A + A49G + L79A + N74T + A75S) VH amino acid sequenceADWA11VH5 D61E  40 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVR VH amino acidQAPGKGLEWVGWIDPDNGNTIYEPKFQGRFTISADTSK sequenceNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S ADWA11VH5 N55Q  41EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVR VH amino acidQAPGKGLEWVGWIDPDQGNTIYDPKFQGRFTISADTSK sequenceNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S ADWA11VH5 N28Q  42EVQLVESGGGLVQPGGSLRLSCAASGFQIKDYYMNWVR VH amino acidQAPGKGLEWVGWIDPDQGNTIYDPKFQGRFTISADTSK sequenceNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S ADWA11VH5 K30A  43EVQLVESGGGLVQPGGSLRLSCAASGFNIADYYMNWVR VH amino acidQAPGKGLEWVGWIDPDNGNTIYDPKFQGRFTISADTSK sequenceNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S ADWA11VH5 N57Q  44EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVR VH amino acidQAPGKGLEWVGWIDPDNGQTIYDPKFQGRFTISADTSK sequenceNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S ADWA11VH5 P62A  45EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVR VH amino acidQAPGKGLEWVGWIDPDNGNTIYDAKFQGRFTISADTSK sequenceNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S ADWA11VH5 K63A  46EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVR VH amino acidQAPGKGLEWVGWIDPDNGNTIYDPAFQGRFTISADTSK sequenceNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S Exemplary human  77MLLGTLLLILYILMLCRMFLLVGAPKANTTQPGIVEGG integrin subunitQVLKCDWSSTRRCQPIEFDATGNRDYAKDDPLEFKSHQ alpha-V (ITGAV)WFGASVRSKQDKILACAPLYHWRTEMKQEREPVGTCFL amino acid sequenceQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDGKNMSSLYNFTGEQMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQRASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKKGIVYIFNGRSTGLNAVPSQILEGQWAARSMPPSFGYSMKGATDIDKNGYPDLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKVSCFNVRFCLKADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPSHSKNMTISRGGLMQCEELIAYLRDESEFRDKLTPITIFMEYRLDYRTAADTTGLQPILNQFTPANISRQAHILLDCGEDNVCKPKLEVSVDSDQKKIYIGDDNPLTLIVKAQNQGEGAYEAELIVSIPLQADFIGVVRNNEALARLSCAFKTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLQIQSSNLFDKVSPVVSHKVDLAVLAAVEIRGVSSPDHIFLPIPNWEHKENPETEEDVGPVVQHIYELRNNGPSSFSKAMLHLQWPYKYNNNTLLYILHYDIDGPMNCTSDMEINPLRIKISSLQTTEKNDTVAGQGERDHLITKRDLALSEGDIHTLGCGVAQCLKIVCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNVIEFPYKNLPIEDITNSTLVTTNVTWGIQPAPMPVPVWVIILAVLAGLLLLAVLVFVMYRMGFFKRVRPPQEEQERE QLQPHENGEGNSET Exemplary human 78 MCGSALAFFTAAFVCLQNDRRGPASFLWAAWVFSLVLG integrin subunitLGQGEDNRCASSNAASCARCLALGPECGWCVQEDFISG beta 8 (ITGB8) aminoGSRSERCDIVSNLISKGCSVDSIEYPSVHVIIPTENEI acid sequenceNTQVTPGEVSIQLRPGAEANFMLKVHPLKKYPVDLYYLVDVSASMHNNIEKLNSVGNDLSRKMAFFSRDFRLGFGSYVDKTVSPYISIHPERIHNQCSDYNLDCMPPHGYIHVLSLTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQTSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLGQLSEKLIDNNINVIFAVQGKQFHWYKDLLPLLPGTIAGEIESKAANLNNLVVEAYQKLISEVKVQVENQVQGIYFNITAICPDGSRKPGMEGCRNVTSNDEVLFNVTVTMKKCDVTGGKNYAIIKPIGFNETAKIHIHRNCSCQCEDNRGPKGKCVDETFLDSKCFQCDENKCHFDEDQFSSESCKSHKDQPVCSGRGVCVCGKCSCHKIKLGKVYGKYCEKDDFSCPYHHGNLCAGHGECEAGRCQCFSGWEGDRCQCPSAAAQHCVNSKGQVCSGRGTCVCGRCECTDPRSIGRFCEHCPTCYTACKENWNCMQCLHPHNLSQAILDQCKTSCALMEQQHYVDQTSECFSSPSYLRIFFIIFIVTFLIGLLKVLIIRQVILQWNSNKIKSSSDYRVSASKKDKLILQSVCTRAVTYRREKPEEIKMDISKLN AHETFRCNF Exemplary mouse  79MAAPGRLLLRPRPGGLLLLLPGLLLPLADAFNLDVESP integrin subunitAEYAGPEGSYFGFAVDFFEPSTSSRMFLLVGAPKANTT alpha-V (ITGAV)QPGIVEGGQVLKCECSSSRRCQPIEFDSTGNRDYAKDD amino acid sequencePLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQEREPVGTCFLQDGTKTVEYAPCRSKNIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYWQGQLISDQVAEIISKYDPNVYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIEDFVSGVPRAARTLGMVYIYDGKNMSSLHNFTGEQMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQRAVGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKKGLVYIFNGRSTGLNSVPSQILEGQWAAQSMPPSFGYSMKGATDVDRNGYPDLVVGAFGVDRAVLYRARPVVTVNAGLEVYPSILNQDNKICPLPGTALKVSCFNVRFCLKADGKGTLPRKLHFQVELLLDKLKQKGAIRRALFLHNRSPVHSKTMTVFRGGQMQCEELVAYLRDESEFRDKLTPITIFMEYRLDQRTAADATGLQPILNQFTPANVSRQAHILLDCGEDNVCKPKLEVSVNSDQKKIYIGDDNPLTLTVKAQNQGEGAYEAELIVSIPPQADFIGVVRNNEALARLSCAFKTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLKIQSSNSFDNVSPVVSYKVDLAVLAAVEIRGVSSPDHIFLPIPNWEYKENPETEEDVGPIVQHIYELRNNGPSSFSKAILNLQWPYKYNNNTLLYILHYDIDGPMNCTADTEINPLRIKTPEKNDTAAAGQGERNHLITKRDLTLREGDVHTLGCGIAKCLQITCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNIIEFPYKNLPIEDLFNSTLVITNITWGIQPAPMPVPVWVIILAVLAGLLLLAVLVFVMYRMGFFKRVRPPQEE QEREQLQPHENGEGNSETExemplary mouse  80 MCGSALAFLTAALLSLHNCQRGPALVLGAAWVFSLVLGintegrin subunit LGQSEHNRCGSANVVSCARCLQLGPECGWCVQEDFVSGbeta 8 (ITGB8) amino GSGSERCDTVSSLISKGCPVDSIEYLSVHVVTSSENEIacid sequence NTQVTPGEVSVQLHPGAEANFMLKVRPLKKYPVDLYYLVDVSASMHNNIEKLNSVGNDLSKKMALYSRDFRLGFGSYVDKTVSPYISIHPERIHNQCSDYNLDCMPPHGYIHVLSLTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQTSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLGQLSEKLIDNNINVIFAVQGKQFHWYKDLLPLLPGAIAGEIESKAANLNNLVVEAYKKIISEVKVQLENQVHGVHFNITAICPDGARKPGISGCGNVTSNDEVLFNVTVVMKTCDIMGGKNYAIIKPIGFNETTKVHIHRSCSCQCENHRGLKGQCAEAAPDPKCPQCDDSRCHFDEDQFPSETCKPQEDQPVCSGRGVCICGKCLCHKTKLGRVYGQYCEKDDFSCPYLHGDVCAGHGECEGGRCQCFSGWEGDRCQCPSASAQHCVNSKGQVCSGRGTCVCGRCECTDPRSIGRLCEHCPTCHLSCSENWNCLQCLHPHNLSQAALDQCKSSCAVMEQHRMDQTSECLSGPSYLRIFFIIFIVTFLIGLLKVLIIRQVILQWNNNKIKSSSDYRMSASKKDKLILQSVCTRAVTYRREKPEEIKMDISKLNAQ EAFRCNF Exemplary cynomolgus  84MASPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSP integrin subunitAEYSGPEGSYFGFAVDFFVPSASSRMFLLVGAPKANTT alpha-V (ITGAV)QPGIVEGGQVLKCDWSSTRRCQPIEFDATGNRDYAKDD amino acid sequencePLEFKSHQWFGASVRSKQDKILACAPLYHWRTELKQEREPVGTCFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVLLGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDSYLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDGKNMSSIYNFTGDQMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQRASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKKGIVYIFNGRSTGLNAVPSQILEGQWAARSMPPSFGYSMKGATDIDKNGYPDLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKVSCFNVRFCLKADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPSHSKNMTISRGGLMQCEELIAYLRDESEFRDKLTPITIFMEYWLDYRTAADTTGLQPILNQFTPANISRQAHILLDCGEDNVCKPKLEVFVDSDQKKIYIGDDNPLTLIVKAQNQGEGAYEAELIVSIPLQADFIGVVRNSEALARLSCAFKTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLQIQSSNLFDKVSPVVSHKVDLAVLAAVEIRGVSSPDHIFLPIPNWEHKENPETEEDVGPVVQHIYELRNNGPSSFSKAMLHLQWPYKYNNNTLLYILHYDIDGPMNCTSDMEINPLRIKISSLQATEKNDTVAGQGERDHLITKRDLALSEGDIHTLGCGVAQCLKIVCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNVIEFPYKNLPIEDITNSTLVTTNVTWGIQPAPMPVPVWVIILAVLAGLLLLAVLVFVMYRMGFFKRVRP PQEEQEREQLQPHENGEGNSETExemplary cynomolgus  85 MCGSALAFFTAAFVCLQNDRRGPASFLWAAWVLSLVLGintegrin subunit LGQGEDNICASSNAASCARCLALGPECGWCVQEDFISGbeta 8 (ITGB8) amino GSRSERCDIVSNLISKGCSVDSIEYPSVHVIIPTENEIacid sequence NTQVTPGEVSIQLRPGAEANFMLKIHPLKKYPVDLYYLVDVSASMHNNIEKLNSVGNDLSRKMAFFSRDFRLGFGSYVDKTVSPYISIHPERIHNQCSDYNLDCMPPHGYIHVLSLTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQTSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLGQLSEKLIDNNINVIFAVQGKQFHWYKDLLPLLPGTIAGEIESKAANLNNLVVEAYQKLISEVKVHVENQVQGVYFNITAICPDGSRKPGMEGCRNVTSNHEVLFNVTVTMKKCDVTGGKNYAIIKPIGFNETAKIHIHRNCSCQCEDNRGPKGKCVDETFLDSKCFQCDENKCHFDEDQFSSESCKSHKDQPVCSGRGVCVCGKCSCHKIKLGKVYGKYCEKDDFSCPYHHGNLCAGHGECEAGRCQCFSGWEGDRCQCPSAAAQHCVNSKGQVCSGRGTCVCGRCECTDPRSIGRFCEHCPTCHTACKENWNCVQCLHPHNLSQAILDQCKTSCALMEQQHYVDQTSECFSSPSYLRIFFIIFIVTFLIGLLKVLIIRQVILQWNSNKIKSSSDYRVSASKKDKLILQSVCTRAVTYRREKPEEIKMDISKLN AHETFRCNF Exemplary human wild 81 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT type IgG1 FcVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL (includes portion ofGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA C_(H)1 and hinge, C_(H)2PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE and C_(H)3)DPEVKFNWYVDGVEVHNAKTKPREEQY

YRVVSVLT Wild type LLGG VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREeffector function PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES sequence isNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN indicated in italicsVFSCSVMHEALHNHYTQKSLSLSPG NST Asn297 N-linked glycosylation siteExemplary human IgG1  82 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTconstant region VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL Wild type LLGGGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA effector functionPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE sequence isDPEVKFNWYVDGVEVHNAKTKPREEQY

YRVVSVLT indicated in italics VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRENST Asn297 N-linked PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESglycosylation site NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIncludes terminal VFSCSVMHEALHNHYTQKSLSLSPGK Lysine Exemplary human 184ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT effectorless IgG1VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL constant regionGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGExemplary human 192 GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCeffectorless IgG1 CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGG constant regionGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG nucleic acidGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA sequenceCACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCGGA Exemplary human 181ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT effectorless IgG1VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL constant regionGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA (with terminalPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE lysine)DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKExemplary human 193 GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCeffectorless IgG1 CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGG constant regionGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG nucleic acidGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA sequence (withCACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT terminal lysine)CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCTGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCCGGAA AA Exemplary human IgG2  70ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT constant regionVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKExemplary human  83 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKkappa light chain VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKconstant region (Cκ) ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Exemplary human 194CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCC kappa light chainATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTG constant region (Cκ)TGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA nucleic acidGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA sequenceCTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGTExemplary murine  86 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTIgG1 heavy chain VTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWP constant regionSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPPVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKAFACAVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSV LHEGLHNHHTEKSLSHSPGKExemplary murine  87 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINlight chain constant VKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTK regionDEYERHNSYTCEATHKTSTSPIVKSFNRNEC adwa_VH_1.1 T28N +  88EVQLVESGGGLVQPGGSLRLSCAASGFniKDYYMNWVR F29IQAPGKGLEWVAWIDPDNGNTIYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S adwa_VH_1.2 T28N +  89EVQLVESGGGLVQPGGSLRLSCAASGFniKDYYMNWVR F29I + R72AQAPGKGLEWVAWIDPDNGNTIYDPKFQGRFTISaDNAKNSLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S adwa_VH_1.3 T28N +  90EVQLVESGGGLVQPGGSLRLSCAASGFniKDYYMNWVR F29I + R72A + A49G +QAPGKGLEWVgWIDPDNGNTIYDPKFQGRFTISaDNAK L79ANSaYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S adwa_VH_1.4 T28N +  91EVQLVESGGGLVQPGGSLRLSCAASGFniKDYYMNWVR F29I + R72A + N74T +QAPGKGLEWVAWIDPDNGNTIYDPKFQGRFTISaDtsK A75SNSLYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S adwa_VL_1.2 L46R +  92DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYL Y36FFWfQQKPGKAPKrLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIK VH05-2(F64V) VK01  93EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDQGNTIYEPKVQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVS S 5662_01  94 EPKFQGRFTISADTS5662_02  95 TAVYYCARRLLMDYW 5662_03  96 TAVYYSARRLLXDYW 5662_04  97KSLLHFNGNTYLFWY 5662_05  98 PKRLIYYMSNLASGV 5662_06  99 PKRLIYYXSNLASGV5662_07 100 LIYYMSNLASGVPSR 5662_08 101 LIYYXSNLASGVPSR 5662_09 102FATYYCMQSLEYPFT 5662_10 103 FATYYSXQSLEYPFT 5662_11 104 EYPFTFGQGTKVEIK5662_12 105 EPKVQGRFTISADTS 5662_13 106 KSLSHFNGNTYLFWY 5662_14 107FATYYCQQSLEYPFT 5662_15 108 FATYYSQQSLEYPFT 5662_16 109 FATYYCMQSYEYPFT5662_17 110 FATYYSXQSYEYPFT 5662_18 111 EYPFTFGGGTKVEIK 5662_19 112KRLIYYASNLASGVP 5662_20 113 KRLIYYMSSLASGVP 5662_21 114 KRLIYYXSSLASGVP5662_22 115 QGDSLRTYYASWYQQ 5662_23 116 VLVIYGKHKRPSGIP 5662_24 117EADYYCMSRSIWGNP 5662_25 118 EADYYSXSRSIWGNP 5662_26 119 SETLSLTCAVSGYST5662_27 120 GLEWIGSISHTGNTY 5662_28 121 NPPLKSRVTISVDTS 5662_29 122DTAVVYCARGGGISR ADWA 11 VK01 123 DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGECADWA11 VH05 124 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDNGNTIYDPKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKADWA11 VH05 without 182 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRterminal lysine QAPGKGLEWVGWIDPDNGNTIYDPKFQGRFTISADTSK residueNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGWild type human IgG1 125 EPKSCDKTHTCPPCPAPELLGGP hingeEffector null (3m, 126 EPKSCDKTHTCPPCPAPEAAGAP triple mutant)variant human IgG1 hinge IGHV3-07 (DP-54) 127EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVR heavy chain germlineQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAK NSLYLQMNSLRAEDTAVYYCARIGKV1-39 (DPK-9) 128 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQlight chain germline KPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP synthetic peptide HA 129 PKYVKQNTLKLAT derived fromInfluenza A hemagglutinin TET 830 modified/T- 130 AQYIKANSKFIGITELhelper epitope from tetanus toxoid IMGT - heavy chain 195EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYWMSWVRQA PGKGLEWVAN IKQDGSEKYYVDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR IMGT - light chain 196DIQMTQSPSS LSASVGDRVT ITCRASQSIS ----S-YLNW YQQKPGKAPK LLIYAASSLQSGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCQQSYSTP *In some peptides,methionine was replaced with norleucine.

TABLE 14 Exemplary heavy chain CDRs according to Kabat VH (SEQ ID NO)CDR-H1 (SEQ ID NO) CDR-H2 (SEQ ID NO) CDR-H3 (SEQ ID NO)Mouse ADWA-11 VH DYYMN (SEQ ID NO: WIDPDNGNTIYDPKF RLLMDY (SEQ ID NO:(SEQ ID NO: 20) 22) QG (SEQ ID NO: 23) 24) ADWA11 2.4 VHDYYMN (SEQ ID NO: WIDPDQGNTIYEPKF RLLMDY (SEQ ID NO: (SEQ ID NO: 6) 8)QG (SEQ ID NO: 9) 10) ADWA11VH1 DYYMN (SEQ ID NO: WIDPDNGNTIYDQKFRLLMDY (SEQ ID NO: IGHV1-46 (SEQ ID 8) QG (SEQ ID NO: 157) 10) NO: 34)ADWA11VH2 DYYMN (SEQ ID NO: WIDPDNGNTIYDDSV RLLMDY (SEQ ID NO:IGHV3-23 (SEQ ID 8) KG (SEQ ID NO: 158) 10) NO: 35) ADWA11VH3DYYMN (SEQ ID NO: WIDPDNGNTIYDDSV RLLMDY (SEQ ID NO: IGHV3-30 (SEQ ID 8)KG (SEQ ID NO: 158) 10) NO: 36) ADWA11VH4 DYYMN (SEQ ID NO:WIDPDNGNTIYDQKF RLLMDY (SEQ ID NO: IGHV1-69 (SEQ ID 8)QG (SEQ ID NO: 157) 10) NO: 37) ADWA11VH5 DYYMN (SEQ ID NO:WIDPDNGNTIYDDSV RLLMDY (SEQ ID NO: IGHV3-48 (SEQ ID 8)KG (SEQ ID NO: 158) 10) NO: 38) ADWA11 VH05_VK1 DYYMN (SEQ ID NO:WIDPDNGNTIYDPKF RLLMDY (SEQ ID NO: (also referred to 8)QG (SEQ ID NO: 23) 10) herein as adwa_VH_1.5 T28N + F29I + R72A +A49G + L79A +0 N74T + A75S) (SEQ ID NO: 39) ADWA11VH5 D61EDYYMN (SEQ ID NO: WIDPDNGNTIYEPKF RLLMDY (SEQ ID NO: (SEQ ID NO: 40) 8)QG (SEQ ID NO: 160) 10) ADWA11VH5 N55Q DYYMN (SEQ ID NO: WIDPDQGNTIYDPKFRLLMDY (SEQ ID NO: (SEQ ID NO: 41) 8) QG (SEQ ID NO: 161) 10)ADWA11VH5 N28Q DYYMN (SEQ ID NO: WIDPDQGNTIYDPKF RLLMDY (SEQ ID NO:(SEQ ID NO: 42) 8) QG (SEQ ID NO: 161) 10) ADWA11VH5 K30ADYYMN (SEQ ID NO: WIDPDNGNTIYDPKF RLLMDY (SEQ ID NO: (SEQ ID NO: 43) 8)QG (SEQ ID NO: 23) 10) ADWA11VH5 N57Q DYYMN (SEQ ID NO: WIDPDNGQTIYDPKFRLLMDY (SEQ ID NO: (SEQ ID NO: 44) 8) QG (SEQ ID NO: 162) 10)ADWA11VH5 P62A DYYMN (SEQ ID NO: WIDPDNGNTIYDAKF RLLMDY (SEQ ID NO:(SEQ ID NO: 45) 8) QG (SEQ ID NO: 163) 10) ADWA11VH5 K63ADYYMN (SEQ ID NO: WIDPDNGNTIYDPAF RLLMDY (SEQ ID NO: (SEQ ID NO: 46) 8)QG (SEQ ID NO: 165) 10) adwa_VH_1.1 DYYMN (SEQ ID NO: WIDPDNGNTIYDPKFRLLMDY (SEQ ID NO: T28N + F29I (SEQ ID 8) QG (SEQ ID NO: 23) 10) NO: 88)adwa_VH_1.2 DYYMN (SEQ ID NO: WIDPDNGNTIYDPKF RLLMDY (SEQ ID NO:T28N + F29I + R72A 8) QG (SEQ ID NO: 23) 10) (SEQ ID NO: 89) adwa_VH_1.3DYYMN (SEQ ID NO: WIDPDNGNTIYDPKF RLLMDY (SEQ ID NO:T28N + F29I + R72A + 8) QG (SEQ ID NO: 23) 10) A49G + L79A (SEQID NO: 90) adwa_VH_1.4 DYYMN (SEQ ID NO: WIDPDNGNTIYDPKFRLLMDY (SEQ ID NO: T28N + F29I + R72A + 8) QG (SEQ ID NO: 23) 10)N74T + A75S (SEQ ID NO: 91) VH05-2(F64V) VK01 DYYMN (SEQ ID NO:WIDPDQGNTIYEPKV RLLMDY (SEQ ID NO: (SEQ ID NO: 93) 8)QG (SEQ ID NO: 166) 10)

TABLE 15 Exemplary light chain CDRs according to Kabat VL (SEQ ID NO)CDR-L1 (SEQ ID NO) CDR-L2 (SEQ ID NO) CDR-L3 (SEQ ID NO)Mouse ADWA-11 VL RSTKSLLHFNGNTYL YYMSNLAS (SEQ ID MQSLEYPFT (SEQ ID(SEQ ID NO: 21) F (SEQ ID NO: 25) NO: 26) NO: 27) ADWA11 2.4 (SEQRSTKSLSHFNGNTYL YYMSSLAS (SEQ ID QQSLEYPFT (SEQ ID ID NO: 7)F (SEQ ID NO: 11) NO: 12) NO: 13) ADWA11_VK01 (1) RSTKSLLHFNGNTYLYYMSNLAS (SEQ ID MQSLEYPFT (SEQ ID (also referred to F (SEQ ID NO: 25)NO: 26) NO: 27) herein as adwa_VL_1.1 L46R) (SEQ ID NO: 47)ADWA11_VK01_1a RSTKSILHFNGNTYLF YYMSNLAS (SEQ ID MQSLEYPFT (SEQ ID(1) L29I (SEQ ID NO: (SEQ ID NO: 138) NO: 26) NO: 27) 48) ADWA11_VK01_1bRSTKSLSHFNGNTYL YYMSNLAS (SEQ ID MQSLEYPFT (SEQ ID (1) L305 (SEQ IDF (SEQ ID NO: 11) NO: 26) NO: 27) NO: 49) ADWA11_VK01_1c RSTKSLLHFNGNSYLYYMSNLAS (SEQ ID MQSLEYPFT (SEQ ID (1) T365 (SEQ ID F (SEQ ID NO: 140)NO: 26) NO: 27) NO: 50) ADWA11_VK01_2a RSTKSLLHFNGNTYL YAMSNLAS (SEQ IDMQSLEYPFT (SEQ ID (1) Y55A (SEQ ID F (SEQ ID NO: 25) NO: 142) NO: 27)NO: 51) ADWA11_VK01_2b RSTKSLLHFNGNTYL YYASNLAS (SEQ IDMQSLEYPFT (SEQ ID (1) M56A (SEQ ID F (SEQ ID NO: 25) NO: 144) NO: 27)NO: 52) ADWA11_VK01_2c RSTKSLLHFNGNTYL YYMSSLAS (SEQ IDMQSLEYPFT (SEQ ID (1) N58S (SEQ ID F (SEQ ID NO: 25) NO: 12) NO: 27)NO: 53) ADWA11_VK01_2d RSTKSLLHFNGNTYL YYMSNLQS (SEQ IDMQSLEYPFT (SEQ ID (1) A60Q (SEQ ID F (SEQ ID NO: 25) NO: 146) NO: 27)NO: 54) ADWA11_VK01_3a RSTKSLLHFNGNTYL YYMSNLAS (SEQ IDQQSLEYPFT (SEQ ID (1) M94Q (SEQ ID F (SEQ ID NO: 25) NO: 26) NO: 13)NO: 55) ADWA11_VK01_3b RSTKSLLHFNGNTYL YYMSNLAS (SEQ IDMQSYEYPFT (SEQ ID (1) L97Y (SEQ ID F (SEQ ID NO: 25) NO: 26) NO: 147)NO: 56) ADWA11_VK01_3c RSTKSLLHFNGNTYL YYMSNLAS (SEQ IDMQSLSYPFT (SEQ ID (1) E98S (SEQ ID F (SEQ ID NO: 25) NO: 26) NO: 149)NO: 57) ADWA11_VK01_3d RSTKSLLHFNGNTYL YYMSNLAS (SEQ IDMQSLETPFT (SEQ ID (1) Y99T (SEQ ID F (SEQ ID NO: 25) NO: 26) NO: 151)NO: 58) ADWA11_VK01_4a RSTKSLLHFNGNTYL YYMSNLAS (SEQ IDMQSLEYPLT (SEQ ID (1) F101L (SEQ ID F (SEQ ID NO: 25) NO: 26) NO: 153)NO: 59) ADWA11_VK01_4b RSTKSLLHFNGNTYL YYMSNLAS (SEQ IDMQSLEYPWT (SEQ ID (1) F101W (SEQ ID F (SEQ ID NO: 25) NO: 26) NO: 155)NO: 60) ADWA11_VK01_4c RSTKSLLHFNGNTYL YYMSNLAS (SEQ IDMQSLEYPFT (SEQ ID (1) Q105G (SEQ ID F (SEQ ID NO: 25) NO: 26) NO: 27)NO: 61) ADWA11VK1 RSTKSLLHFNGNTYL YYMSNLAS (SEQ ID MQSLEYPFT (SEQ IDIGKV2-28 (SEQ ID F (SEQ ID NO: 25) NO: 26) NO: 27) NO: 62) ADWA11VK2RSTKSLLHFNGNTYL YYMSNLAS (SEQ ID MQSLEYPFT (SEQ ID IGKV2-30 (SEQ IDF (SEQ ID NO: 25) NO: 26) NO: 27) NO: 63) ADWA11VK3 RSTKSLLHFNGNTYLYYMSNLAS (SEQ ID MQSLEYPFT (SEQ ID IGKV4-1 (SEQ ID F (SEQ ID NO: 25)NO: 26) NO: 27) NO: 64) ADWA11VK4 RSTKSLLHFNGNTYL YYMSNLAS (SEQ IDMQSLEYPFT (SEQ ID IGKV1-39 (SEQ ID F (SEQ ID NO: 25) NO: 26) NO: 27)NO: 65) ADWA11VK5 RSTKSLLHFNGNTYL YYMSNLAS (SEQ ID MQSLEYPFT (SEQ IDIGKV3-11 (SEQ ID F (SEQ ID NO: 25) NO: 26) NO: 27) NO: 66)ADWA11_VK01_2.1 RSTKSLSHFNGNTYL YYMSNLAS (SEQ ID QQSLEYPFT (SEQ ID(SEQ ID NO: 67) F (SEQ ID NO: 11) NO: 26) NO: 13) ADWA11_VK012.2RSTKSLSHFNGNTYL YYMSNLAS (SEQ ID MQSYEYPFT (SEQ ID (SEQ ID NO: 68)F (SEQ ID NO: 11) NO: 26) NO: 147) ADWA11_VK01_2.3 RSTKSLSHFNGNTYLYYASNLAS (SEQ ID QQSLEYPFT (SEQ ID (SEQ ID NO: 69) F (SEQ ID NO: 11)NO: 144) NO: 13) adwa_VL_1.2 L46R + RSTKSLLHFNGNTYL YYMSNLAS (SEQ IDMQSLEYPFT (SEQ ID Y36F (SEQ ID NO: F (SEQ ID NO: 25) NO: 26) NO: 27) 92)

TABLE 16 Exemplary heavy chain CDRs according to Chothia VH (SEQ ID NO)CDR-H1 (SEQ ID NO) CDR-H2 (SEQ ID NO) CDR-H3 (SEQ ID NO) Mouse ADWA-11GFNIKDYYMN (SEQ WIDPDNGN (SEQ ID RLLMDY (SEQ ID VH (SEQ ID NO: 20)ID NO: 28) NO: 29) NO: 30) ADWA11 2.4 VH GFNIKDYYMN (SEQWIDPDQGN (SEQ ID RLLMDY (SEQ ID (SEQ ID NO: 6) ID NO: 14) NO: 15)NO: 16) ADWA11VH1 GFNIKDYYMN (SEQ WIDPDNGN (SEQ ID RLLMDY (SEQ IDIGHV1-46 (SEQ ID ID NO: 14) NO: 29) NO: 16) NO: 34) ADWA11VH2GFNIKDYYMN (SEQ WIDPDNGN (SEQ ID RLLMDY (SEQ ID IGHV3-23 (SEQ IDID NO: 14) NO: 29) NO: 16) NO: 35) ADWA11VH3 GFNIKDYYMN (SEQWIDPDNGN (SEQ ID RLLMDY (SEQ ID IGHV3-30 (SEQ ID ID NO: 14) NO: 29)NO: 16) NO: 36) ADWA11VH4 GFNIKDYYMN (SEQ WIDPDNGN (SEQ IDRLLMDY (SEQ ID IGHV1-69 (SEQ ID ID NO: 14) NO: 29) NO: 16) NO: 37)ADWA11VH5 GFNIKDYYMN (SEQ WIDPDNGN (SEQ ID RLLMDY (SEQ IDIGHV3-48 (SEQ ID ID NO: 14) NO: 29) NO: 16) NO: 38) ADWA11GFNIKDYYMN (SEQ WIDPDNGN (SEQ ID RLLMDY (SEQ ID VH05_VK1 (alsoID NO: 14) NO: 29) NO: 16) referred to herein as adwa_VH_1.5T28N + F29I + R72A + A49G + L79A + N74T + A75S) (SEQ ID NO: 39)ADWA11VH5 D61E GFNIKDYYMN (SEQ WIDPDNGN (SEQ ID RLLMDY (SEQ ID(SEQ ID NO: 40) ID NO: 14) NO: 29) NO: 16) ADWA11VH5 N55QGFNIKDYYMN (SEQ WIDPDQGN (SEQ ID RLLMDY (SEQ ID (SEQ ID NO: 41)ID NO: 14) NO: 15) NO: 16) ADWA11VH5 N28Q GFNIKDYYMN (SEQWIDPDQGN (SEQ ID RLLMDY (SEQ ID (SEQ ID NO: 42) ID NO: 14) NO: 15)NO: 16) ADWA11VH5 K30A GFNIADYYMN (SEQ WIDPDNGN (SEQ ID RLLMDY (SEQ ID(SEQ ID NO: 43) ID NO: 159) NO: 29) NO: 16) ADWA11VH5 N57QGFNIKDYYMN (SEQ WIDPDNGQ (SEQ ID RLLMDY (SEQ ID (SEQ ID NO: 44)ID NO: 14) NO: 164) NO: 16) ADWA11VH5 P62A GFNIKDYYMN (SEQWIDPDNGN (SEQ ID RLLMDY (SEQ ID (SEQ ID NO: 45) ID NO: 14) NO: 29)NO: 16) ADWA11VH5 K63A GFNIKDYYMN (SEQ WIDPDNGN (SEQ ID RLLMDY (SEQ ID(SEQ ID NO: 46) ID NO: 14) NO: 29) NO: 16) adwa_VH_1.1 GFNIKDYYMN (SEQWIDPDNGN (SEQ ID RLLMDY (SEQ ID T28N + F29I (SEQ ID NO: 14) NO: 29)NO: 16) ID NO: 88) adwa_VH_1.2 GFNIKDYYMN (SEQ WIDPDNGN (SEQ IDRLLMDY (SEQ ID T28N + F29I + R72A ID NO: 14) NO: 29) NO: 16)(SEQ ID NO: 89) adwa_VH_1.3 GFNIKDYYMN (SEQ WIDPDNGN (SEQ IDRLLMDY (SEQ ID T28N + F29I + R72A + ID NO: 14) NO: 29) NO: 16)A49G + L79A (SEQ ID NO: 90) adwa_VH_1.4 GFNIKDYYMN (SEQ WIDPDNGN (SEQ IDRLLMDY (SEQ ID T28N + F29I + R72A + ID NO: 14) NO: 29) NO: 16)N74T + A75S (SEQ ID NO: 91) VH05-2(F64V) VK01 GFNIKDYYMN (SEQWIDPDQGN (SEQ ID RLLMDY (SEQ ID (SEQ ID NO: 93) ID NO: 14) NO: 15)NO: 16)

TABLE 17 Exemplary light chain CDRs according to Chothia VL (SEQ ID NO)CDR-L1 (SEQ ID NO) CDR-L2 (SEQ ID NO) CDR-L3 (SEQ ID NO) Mouse ADWA-11STKSLLHFNGNTYL YYMSN (SEQ ID NO: QSLEYPFT (SEQ ID VL (SEQ ID NO: 21) (SEQ ID NO: 31) 32) NO: 33) ADWA11 2.4 (SEQ STKSLSHFNGNTYLYYMSS (SEQ ID NO: QSLEYPFT (SEQ ID ID NO: 7) (SEQ ID NO: 17) 18) NO: 19)ADWA11_VK01 (1) STKSLLHFNGNTYL YYMSN (SEQ ID NO: QSLEYPFT (SEQ ID(also referred to (SEQ ID NO: 31) 32) NO: 19) herein as adwa_VL_1.1L46R) (47) ADWA11_VK01_1a STKSILFIFNGNTYL YYMSN (SEQ ID NO:QSLEYPFT (SEQ ID (1) L29I (SEQ ID (SEQ ID NO: 139) 32) NO: 19) NO: 48)ADWA11_VK01_1b STKSLSHFNGNTYL YYMSN (SEQ ID NO: QSLEYPFT (SEQ ID(1) L305 (SEQ ID (SEQ ID NO: 17) 32) NO: 19) NO: 49) ADWA11_VK01_1cSTKSLLHFNGNSYL YYMSN (SEQ ID NO: QSLEYPFT (SEQ ID (1) T365 (SEQ ID(SEQ ID NO: 141) 32) NO: 19) NO: 50) ADWA11_VK01_2a STKSLLHFNGNTYLYAMSN (SEQ ID NO: QSLEYPFT (SEQ ID (1) Y55A (SEQ ID (SEQ ID NO: 31) 143)NO: 19) NO: 51) ADWA11_VK01_2b STKSLLHFNGNTYL YYASN (SEQ ID NO:QSLEYPFT (SEQ ID (1) M56A (SEQ ID (SEQ ID NO: 31) 145) NO: 19) NO: 52)ADWA11_VK01_2c STKSLLHFNGNTYL YYMSS (SEQ ID NO: QSLEYPFT (SEQ ID(1) N58S (SEQ ID (SEQ ID NO: 31) 18) NO: 19) NO: 53) ADWA11_VK01_2dSTKSLLHFNGNTYL YYMSN (SEQ ID NO: QSLEYPFT (SEQ ID (1) A60Q (SEQ ID(SEQ ID NO: 31) 32) NO: 19) NO: 54) ADWA11_VK01_3a STKSLLHFNGNTYLYYMSN (SEQ ID NO: QSLEYPFT (SEQ ID (1) M94Q (SEQ ID (SEQ ID NO: 31) 32)NO: 19) NO: 55) ADWA11_VK01_3b STKSLLHFNGNTYL YYMSN (SEQ ID NO:QSYEYPFT (SEQ ID (1) L97Y (SEQ ID (SEQ ID NO: 31) 32) NO: 148) NO: 56)ADWA11_VK01_3c STKSLLHFNGNTYL YYMSN (SEQ ID NO: QSLSYPFT (SEQ ID(1) E98S (SEQ ID (SEQ ID NO: 31) 32) NO: 150) NO: 57) ADWA11_VK01_3dSTKSLLHFNGNTYL YYMSN (SEQ ID NO: QSLETPFT (SEQ ID (1) Y99T (SEQ ID(SEQ ID NO: 31) 32) NO: 152) NO: 58) ADWA11_VK01_4a STKSLLHFNGNTYLYYMSN (SEQ ID NO: QSLEYPLT (SEQ ID (1) F101L (SEQ ID (SEQ ID NO: 31) 32)NO: 154) NO: 59) ADWA11_VK01_4b STKSLLHFNGNTYL YYMSN (SEQ ID NO:QSLEYPWT (SEQ ID (1) F101W (SEQ ID  (SEQ ID NO: 31) 32) NO: 156) NO: 60)ADWA11_VK01_4c STKSLLHFNGNTYL YYMSN (SEQ ID NO: QSLEYPFT (SEQ ID(1) Q105G (SEQ ID  (SEQ ID NO: 31) 32) NO: 19) NO: 61) ADWA11VK1STKSLLHFNGNTYL YYMSN (SEQ ID NO: QSLEYPFT (SEQ ID IGKV2-28 (SEQ ID(SEQ ID NO: 31) 32) NO: 19) NO: 62) ADWA11VK2 STKSLLHFNGNTYLYYMSN (SEQ ID NO: QSLEYPFT (SEQ ID IGKV2-30 (SEQ ID (SEQ ID NO: 31) 32)NO: 19) NO: 63) ADWA11VK3 STKSLLHFNGNTYL YYMSN (SEQ ID NO:QSLEYPFT (SEQ ID IGKV4-1 (SEQ ID (SEQ ID NO: 31) 32) NO: 19) NO: 64)ADWA11VK4 STKSLLHFNGNTYL YYMSN (SEQ ID NO: QSLEYPFT (SEQ IDIGKV1-39 (SEQ ID (SEQ ID NO: 31) 32) NO: 19) NO: 65) ADWA11VK5STKSLLHFNGNTYL YYMSN (SEQ ID NO: QSLEYPFT (SEQ ID IGKV3-11 (SEQ ID(SEQ ID NO: 31) 32) NO: 19) NO: 66) ADWA11_VK01_2.1 STKSLSHFNGNTYLYYMSN (SEQ ID NO: QSLEYPFT (SEQ ID (SEQ ID NO: 67) (SEQ ID NO: 17) 32)NO: 19) ADWA11_VK012.2 STKSLSHFNGNTYL YYMSN (SEQ ID NO: QSYEYPFT (SEQ ID(SEQ ID NO: 68) (SEQ ID NO: 17) 32) NO: 148) ADWA11_VK012.3STKSLSHFNGNTYL YYASN (SEQ ID NO: QSLEYPFT (SEQ ID (SEQ ID NO: 69)(SEQ ID NO: 17) 145) NO: 19) adwa_VL_1.2 STKSLLHFNGNTYLYYMSN (SEQ ID NO: QSLEYPFT (SEQ ID L46R + Y36F (SEQ (SEQ ID NO: 31) 32)NO: 19) ID NO: 92)

In some aspects, the antibody, or antigen-binding fragment thereof,comprises a CDR-L1, a CDR-L2, and a CDR-L3 as set forth in the aminoacid sequence of at least one of SEQ ID NOs: 11-13, 17-19, 25-27, 31-33,or 71-76.

In some aspects, the antibody, or antigen-binding fragment thereof,further comprises a CDR-H1, a CDR-H2, and a CDR-H3 as set forth in theamino acid sequence of at least one of SEQ ID NOs: 8-10, 14-16, 22-24,or 28-30.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1, a CDR-L2, a CDR-L3 as set forth in the amino acidsequence of SEQ ID NO: 7, and a CDR-H1, a CDR-H2, and a CDR-H3 as setforth in the amino acid sequence of SEQ ID NO: 6.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1, a CDR-L2, a CDR-L3 as set forth in the amino acidsequence of SEQ ID NO: 7, and a CDR-H1, a CDR-H2, and a CDR-H3 as setforth in the amino acid sequence of SEQ ID NO: 20.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1, a CDR-L2, a CDR-L3 as set forth in the amino acidsequence of SEQ ID NO: 7, and a CDR-H1, a CDR-H2, and a CDR-H3 as setforth in the amino acid sequence of any one of SEQ ID NOs: 34-46, 88-91or 93.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1, a CDR-L2, a CDR-L3 as set forth in the amino acidsequence of SEQ ID NO: 21, and a CDR-H1, a CDR-H2, and a CDR-H3 as setforth in the amino acid sequence of SEQ ID NO: 6.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1, a CDR-L2, a CDR-L3 as set forth in the amino acidsequence of any one of SEQ ID NOs: 47-69 or 92, and a CDR-H1, a CDR-H2,and a CDR-H3 as set forth in the amino acid sequence of SEQ ID NO: 6.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1, a CDR-L2, a CDR-L3 as set forth in the amino acidsequence of SEQ ID NO: 5, and a CDR-H1, a CDR-H2, and a CDR-H3 as setforth in the amino acid sequence of SEQ ID NO: 2.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1, a CDR-L2, a CDR-L3 as set forth in the amino acidsequence of SEQ ID NO: 5, and a CDR-H1, a CDR-H2, and a CDR-H3 as setforth in the amino acid sequence of SEQ ID NO: 3.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1, a CDR-L2, a CDR-L3 as set forth in the amino acidsequence of SEQ ID NO: 5, and a CDR-H1, a CDR-H2, and a CDR-H3 as setforth in the amino acid sequence of SEQ ID NO: 124 or SEQ ID NO: 182.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1, a CDR-L2, a CDR-L3 as set forth in the amino acidsequence of SEQ ID NO: 123, and a CDR-H1, a CDR-H2, and a CDR-H3 as setforth in the amino acid sequence of SEQ ID NO: 124 or SEQ ID NO: 182.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1, a CDR-L2, a CDR-L3 as set forth in the amino acidsequence of SEQ ID NO: 123, and a CDR-H1, a CDR-H2, and a CDR-H3 as setforth in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

In some aspects, the antibody, or antigen-binding fragment thereof,comprises a CDR-L1, a CDR-L2, and a CDR-L3 as set forth in the aminoacid sequence encoded by the insert of the plasmid deposited with theATCC having the Accession number PTA-124918.

In some aspects, the antibody, or antigen-binding fragment thereof,comprises a CDR-H1, a CDR-H2, and a CDR-H3 as set forth in the aminoacid sequence encoded by the insert of the plasmid deposited with theATCC having Accession number PTA-124917.

In some aspects, the antibody, or antigen-binding fragment thereof,comprises a CDR-L1, a CDR-L2, and a CDR-L3 amino acid sequence encodedby the insert of the plasmid deposited with the ATCC having theAccession number Accession number PTA-124918, and a CDR-H1, a CDR-H2,and a CDR-H3 amino acid sequence encoded by the insert of the plasmiddeposited with the ATCC having Accession number PTA-124917.

In some aspects, the antibody, or antigen-binding fragment thereof,comprises a light chain variable region comprising the amino acidsequence encoded by the insert of the plasmid deposited with the ATCChaving the Accession number PTA-124918.

In some aspects, the antibody, or antigen-binding fragment thereof,comprises a heavy chain variable region comprising the amino acidsequence encoded by the insert of the plasmid deposited with the ATCChaving the Accession number PTA-124917.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:11, aCDR-L2 comprising the amino acid sequence of SEQ ID NO:12, a CDR-L3comprising the amino acid sequence of SEQ ID NO:13, a CDR-H1 comprisingthe amino acid sequence of SEQ ID NO:8, a CDR-H2 comprising the aminoacid sequence of SEQ ID NO:9, and a CDR-H3 comprising the amino acidsequence of SEQ ID NO:10.

In some aspects, the antibody, or antigen binding fragment thereof,comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:17, aCDR-L2 comprising the amino acid sequence of SEQ ID NO:18, a CDR-L3comprising the amino acid sequence of SEQ ID NO:19, a CDR-H1 comprisingthe amino acid sequence of SEQ ID NO:14, a CDR-H2 comprising the aminoacid sequence of SEQ ID NO:15, and a CDR-H3 comprising the amino acidsequence of SEQ ID NO:16.

In some aspects, an antibody, or antigen-binding fragment thereof, maycomprise a VH comprising an amino acid sequence at least 90% identicalto the amino acid sequence of any one of SEQ ID NOs: 6, 34-46, 88-91 and93 (e.g., SEQ ID NO: 6). The VH may comprise an amino acid sequence atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to theamino acid sequence of any one of SEQ ID NOs: 6, 34-46, 88-91 and 93(e.g., SEQ ID NO: 6). The VH may comprise the amino acid sequence of anyone of SEQ ID NOs: 6, 34-46, 88-91 and 93 (e.g., SEQ ID NO: 6).

In some aspects, an antibody, or antigen-binding fragment thereof, maycomprise a VH comprising an amino acid sequence at least 90% identicalto the amino acid sequence of SEQ ID NO: 6. The VH may comprise an aminoacid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,identical to the amino acid sequence of SEQ ID NO: 6. The VH maycomprise the amino acid sequence of SEQ ID NO: 6. In some embodiments,an antibody, or antigen-binding fragment thereof, comprises a VHcomprising the amino acid sequence of SEQ ID NO: 6. In some embodiments,an antibody, or antigen-binding fragment thereof, comprises a VHconsisting of the amino acid sequence of SEQ ID NO: 6.

In some aspects, an antibody, or antigen-binding fragment thereof, maycomprise a VL comprising an amino acid sequence at least 90% identicalto the amino acid sequence of any one of SEQ ID NOs: 7, 47-69 and 92(e.g., SEQ ID NO: 7). The VL may comprise an amino acid sequence atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to theamino acid sequence of any one of SEQ ID NOs: 7, 47-69 and 92 (e.g., SEQID NO: 7). The VL may comprise the amino acid sequence of any one of SEQID NOs: 7, 47-69 and 92 (e.g., SEQ ID NO: 7).

In some aspects, an antibody, or antigen-binding fragment thereof, maycomprise a VL comprising an amino acid sequence at least 90% identicalto the amino acid sequence of SEQ ID NO: 7. The VL may comprise an aminoacid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,identical to the amino acid sequence of SEQ ID NO: 7. The VL maycomprise the amino acid sequence of SEQ ID NO: 7. In some embodiments,an antibody, or antigen-binding fragment thereof, comprises a VLcomprising the amino acid sequence of SEQ ID NO: 7. In some embodiments,an antibody, or antigen-binding fragment thereof, comprises a VLconsisting of the amino acid sequence of SEQ ID NO: 7.

In some aspects, an antibody, or antigen-binding fragment thereof, maycomprise a heavy chain comprising a VH comprising the amino acidsequence of any one of SEQ ID NOs: 6, 34-46, 88-91 and 93 (e.g., SEQ IDNO: 6), and further comprising an IgG1 constant domain (e.g., an IgG1constant domain comprising the amino acid sequence of any one of SEQ IDNO: 81, 82, 181 or 184). In some aspects, an antibody, orantigen-binding fragment, variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 conservative or non-conservativesubstitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 additions and/or deletions to the full length heavy chain. In afurther aspect, a variant shares at least 65%, at least 75%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity with the full length heavy chain,and wherein said antibody or antigen-binding fragment specifically bindsαvβ8 integrin.

In some embodiments, an antibody, or antigen-binding fragment thereof,comprises a heavy chain comprising a VH comprising the amino acidsequence of SEQ ID NO: 6 and further comprises an IgG1 constant domaincomprising the amino acid sequence of SEQ ID NO: 181 or SEQ ID NO: 184.In some embodiments, an antibody, or antigen-binding fragment thereof,comprises a VH consisting of the amino acid sequence of SEQ ID NO: 6 andfurther comprises an IgG1 constant domain consisting of the amino acidsequence of SEQ ID NO: 181 or SEQ ID NO: 184. In some embodiments, theantibody lacks effector function(s). In yet other embodiments, theantibody molecule has a heavy chain constant region chosen from, e.g.,the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1,IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human)heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In anotherembodiment, the antibody molecule has a light chain constant regionchosen from, e.g., the (e.g., human) light chain constant regions ofkappa (e.g., encoded by the amino acid sequence of SEQ ID NO: 83) orlambda. The constant region can be altered, e.g., mutated, to modify theproperties of the antibody (e.g., to increase or decrease one or moreof: Fc receptor binding, antibody glycosylation, the number of cysteineresidues, effector cell function, and/or complement function). In oneembodiment the antibody has: effector function; and can fix complement.In other embodiments the antibody does not; recruit effector cells; orfix complement. In another embodiment, the antibody has reduced or noability to bind an Fc receptor. For example, it is a isotype or subtype,fragment or other mutant, which does not support binding to an Fcreceptor, e.g., it has a mutagenized or deleted Fc receptor bindingregion.

Methods for altering an antibody constant region are known in the art.Antibodies with altered function, e.g. altered affinity for an effectorligand, such as FcR on a cell, or the Cl component of complement can beproduced by replacing at least one amino acid residue in the constantportion of the antibody with a different residue (see e.g., EP 388151A1, U.S. Pat. Nos. 5,624,821 and 5,648,260, the contents of all of whichare hereby incorporated by reference). Similar type of alterations couldbe described which if applied to the murine, or other speciesimmunoglobulin would reduce or eliminate these functions.

In some embodiments, an antibody, or antigen-binding fragment thereof,comprises a light chain comprising a VL comprising the amino acidsequence of any one of SEQ ID NOs: 7, 47-69 and 92 (e.g., SEQ ID NO: 7),and further comprises an kappa constant domain comprising the amino acidsequence of SEQ ID NO: 83. In some embodiments, an antibody, orantigen-binding fragment thereof, comprises a light chain comprising aVL consisting of the amino acid sequence of SEQ ID NO: 7 and furthercomprises a kappa constant domain consisting of the amino acid sequenceof SEQ ID NO: 83.

In some aspects, an antibody, or antigen-binding fragment thereof,comprises a heavy chain (HC) comprising an amino acid sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to theamino acid sequence of SEQ ID NO: 2. In some embodiments, an antibody,or antigen-binding fragment thereof, comprises a HC comprising the aminoacid sequence of SEQ ID NO: 2. In some embodiments, an antibody, orantigen-binding fragment thereof, comprises a HC consisting of the aminoacid sequence of SEQ ID NO: 2. In some embodiments, the antibody lackseffector function(s).

In some aspects, an antibody, or antigen-binding fragment thereof,comprises a heavy chain (HC) comprising an amino acid sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to theamino acid sequence of SEQ ID NO: 3. In some embodiments, an antibody,or antigen-binding fragment thereof, comprises a HC comprising the aminoacid sequence of SEQ ID NO: 3. In some embodiments, an antibody, orantigen-binding fragment thereof, comprises a HC consisting of the aminoacid sequence of SEQ ID NO: 3. In some embodiments, the antibody lackseffector function(s). In some aspects, an antibody, or antigen-bindingfragment thereof, comprises a light chain (LC) comprising an amino acidsequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,identical to SEQ ID NO: 5. In some embodiments, an antibody, orantigen-binding fragment thereof, comprises a LC comprising the aminoacid sequence of SEQ ID NO: 5. In some embodiments, an antibody, orantigen-binding fragment thereof, comprises a LC consisting of the aminoacid sequence of SEQ ID NO: 5. In some embodiments, the antibody lackseffector function.

Germline Substitutions

In certain embodiments, the antibody, or antigen-binding fragmentthereof, comprises the following heavy chain CDR sequences: (i) CDR-H1comprising SEQ ID NO:22, CDR-H2 comprising SEQ ID NO:23, and CDR-H3comprising SEQ ID NO:24; and/or (ii) the following light chain CDRsequences: CDR-L1 comprising SEQ ID NO:25 or 71, CDR-L2 comprising SEQID NO:26 or 72, and CDR-L3 comprising SEQ ID NO:27 or 73. In certainembodiments, the antibody, or antigen-binding fragment thereof,comprises the following heavy chain CDR sequences: (i) CDR-H1 comprisingSEQ ID NO:28, CDR-H2 comprising SEQ ID NO:29, and CDR-H3 comprising SEQID NO:30; and/or (ii) the following light chain CDR sequences: CDR-L1comprising SEQ ID NO:31 or 74, CDR-L2 comprising SEQ ID NO:32 or 75, andCDR-L3 comprising SEQ ID NO:33 or 76. These are mouse CDRs and,preferably, are grafted or otherwise added in the context of a human VHand VL domain. A wide variety of acceptor human germline sequences areavailable and the process for “humanizing” a non-human species antibodyto use in humans will well-known in the art and also discussed elsewhereherein. Therefore, the skilled artisan would appreciate that the abovemouse CDR sequences can be placed in the context of human V domain aminoacid sequences. In doing so, changes to the acceptor human germlinesequences are generally made to preserve antibody binding and otherdesirable characteristics of the original parent (i.e., donor) antibody.Both the CDRs and framework regions (FW) may be engineered as follows.

In certain embodiments, no more than 11, or no more than 10, no morethan 9, no more than 8, no more than 7, no more than 6, no more than 5,no more than 4, no more than 3, no more than 2, or no more than 1substitution is made in CDR-L1, relative to the amino acid sequence ofSEQ ID NO: 25, 31, 71, or 74. In certain embodiments, no more than 6, nomore than 5, no more than 4, no more than 3, no more than 3, no morethan 2, or no more than one substitution is made in CDR-L2, relative tothe amino acid sequence of SEQ ID NO: 26, 32, 72, or 75. In certainembodiments, no more than 8, no more than 7, no more than 6, no morethan 5, no more than 4, no more than 3, no more than 3, no more than 2,or no more than one substitution is made in CDR-L3, relative to theamino acid sequence of SEQ ID NO: 27, 33, 73, 76. In some embodiments,no more than 10, no more than 9, no more than 8, no more than 7, no morethan 6, no more than 5, no more than 4, no more than 3, no more than 2,or no more than 1 substitution is made in CDR-H1, relative to the aminoacid sequence of SEQ ID NO: 22 or 28. In some embodiments, no more thanno more than 17, no more than 16, no more than 15, no more than 14, nomore than 13, no more than 12, no more than 11, or no more than one 10,no more than 9, no more than 8, no more than 7, no more than 6, no morethan 5, no more than 4, no more than 3, no more than 2, or no more than1 substitution is made in CDR-H2, relative to relative to the amino acidsequence of SEQ ID NO: 23 or 29. In some embodiments, no more than 12,no more than 11, or no more than 10, no more than 9, no more than 8, nomore than 7, no more than 6, no more than 5, no more than 4, no morethan 3, no more than 2, or no more than 1 substitution is made inCDR-H3, relative to the amino acid sequence of SEQ ID NO: 24 or 30. Incertain embodiments, the substitution(s) do not change binding affinity(K_(D)) value by more than 1000-fold, more than 100-fold, or 10-fold. Incertain embodiments, the substitution is a conservative substitutionaccording to Table 1.

In certain embodiments, the substitution is human germline substitutionin which a (donor) CDR residue is replaced with the corresponding humangermline (acceptor) residue, to increase the human amino acid contentand potentially reduce immunogenicity of the antibody as described in,e.g., US Patent Application Publication No. 2017/0073395 and Townsend etal., 2015, Proc. Nat. Acad. Sci. USA 112(50):15354-15359).

Methods and libraries for introducing human germline residues inantibody CDRs are described in detail in US Patent ApplicationPublication No. 2017/0073395, and Townsend et al., 2015, Proc. Natl.Acad. Sci. USA. 112(50):15354-15359, and both are herein incorporated byreference in their entirety.

The antibody, or antigen-binding fragment thereof, may comprise a VHframework comprising a human germline VH framework sequence. The VHframework sequence can be from a human VH3 germline, a VH1 germline, aVH5 germline, or a VH4 germline. Preferred human germline heavy chainframeworks are frameworks derived from VH1, VH3, or VH5 germlines. Forexample, VH frameworks from the following germlines may be used:IGHV3-07, IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69, or IGHV3-48 (germlinenames are based on IMGT germline definition). Preferred human germlinelight chain frameworks are frameworks derived from VK or Vλ, germlines.For example, VL frameworks from the following germlines may be used:IGKV1-39, IGKV2-28, IGKV2-30, IGKV4-1, or IGKV3-11 (germline names arebased on IMGT germline definition). Alternatively, or in addition, theframework sequence may be a human germline consensus framework sequence,such as the framework of human Vλ1 consensus sequence, VK1 consensussequence, VK2 consensus sequence, VK3 consensus sequence, VH3 germlineconsensus sequence, VH1 germline consensus sequence, VH5 germlineconsensus sequence, or VH4 germline consensus sequence. Sequences ofhuman germline frameworks are available from various public databases,such as V-base, IMGT, NCBI, or Abysis.

The antibody, or antigen-binding fragment thereof, may comprise a VLframework comprising a human germline VL framework sequence. The VLframework may comprise one or more amino acid substitutions, additions,or deletions, while still retaining functional and structural similaritywith the germline from which it was derived. In some aspects, the VLframework is at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to a human germline VL framework sequence.In some aspects, the antibody, or antigen binding fragment thereof,comprises a VL framework comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 aminoacid substitutions, additions or deletions relative to the humangermline VL framework sequence. In some aspects, the 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid substitutions, additions or deletions are onlyin the framework regions. In some aspects, the % identity is based onsimilarity with VL excluding those portions herein defined as CDRs.

The human germline VL framework may be the framework of DPK9 (IMGT name:IGKV1-39, e.g., SEQ ID NO:128). The human germline VL framework may bethe framework of IGKV2-28. The human germline VL framework may be theframework of DPK18 (IMGT name: IGKV2-30). The human germline VLframework may be the framework of DPK24 (IMGT name: IGKV4-1). The humangermline VL framework may be the framework of HK102_V1 (IMGT name:IGKV1-5). The human germline VL framework may be the framework of Vg_38K(IMGT name: IGKV3-11). The human germline VL framework may be theframework of human Vλ consensus sequence. The human germline VLframework may be the framework of human Vλ1 consensus sequence. Thehuman germline VL framework may be the framework of human Vλ3 consensussequence. The human germline VL framework may be the framework of humanVK consensus sequence. The human germline VL framework may be theframework of human VK1 consensus sequence. The human germline VLframework may be the framework of human VK2 consensus sequence. Thehuman germline VL framework may be the framework of human VK3 consensussequence.

In some aspects, the VL framework is DPK9 (SEQ ID NO: 128). Othersimilar framework regions are also predicted to deliver advantageousantibodies of the invention comprising CDRs of SEQ ID NOs: 11-13 and17-19; and CDRs specified by the following VL amino acid sequences: 7,47-69 and 92, including, e.g., IGKV2-28, IGKV2-30, IGKV4-1, or IGKV3-11,which may comprise 99, 97, 97, 96, 80, 76, 66, 97, 97, 96, 76, and 74%identity respectively to the FW region of DPK-9 and one or fewer aminoacid differences in common structural features (Kabat Numbering) (A)residues directly underneath CDR (Vernier Zone), L2, L4, L35, L36, L46,L47, L48, L49, L64, L66, L68, L69, L71, (B) VH/VL Chain packingResidues: L36, L38, L44, L46, L87 and (C) canonical CDR Structuralsupport residues L2, L48, L64, L71 (see Lo, “Antibody Humanization byCDR Grafting”, (2004) Antibody Engineering, Vol. 248, Methods inMolecular Biology pp 135-159 and O'Brien and Jones, “Humanization ofMonoclonal Antibodies by CDR Grafting”, (2003) Recombinant Antibodiesfor Cancer Therapy, Vol. 207, Methods in Molecular Biology pp 81-100).Particularly preferred are framework regions of IGKV2-28, IGKV2-30,IGKV4-1, or IGKV3-11 sharing 99, 97, 97, 96, 80, 76, 66% identity toDPK9 respectively and have no amino acid differences in these commonstructural features. In some aspects, the % identity is based onsimilarity with VL excluding those portions herein defined as CDRs.

The antibody, or antigen-binding fragment thereof, may comprise a VHframework comprising a human germline VH framework sequence. The VHframework may comprise one or more amino acid substitutions, additions,or deletions, while still retaining functional and structural similaritywith the germline from which it was derived. In some aspects, the VHframework is at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to a human germline VH framework sequence.In some aspects, the antibody, or antigen binding fragment thereof,comprises a VH framework comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 aminoacid substitutions, additions or deletions relative to the humangermline VH framework sequence. In some aspects, the 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid substitutions, additions or deletions are onlyin the framework regions. In some aspects, the % identity is based onsimilarity with VH excluding those portions herein defined as CDRs.

The human germline VH framework may be, for example, the framework ofIGHV3-07 (also known as DP-54), IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69,or IGHV3-48. The human germline VH framework may be the framework ofhuman VH germline consensus sequence. The human germline VH frameworkmay be the framework of human VH3 germline consensus sequence. The humangermline VH framework may be the framework of human VH5 germlineconsensus sequence. The human germline VH framework may be the frameworkof human VH1 germline consensus sequence. The human germline VHframework may be the framework of human VH4 germline consensus sequence.

In some aspects, the VH framework is IGHV3-07 (SEQ ID NO: 127). Othersimilar framework regions are also predicted to deliver advantageousantibodies of the invention comprising CDRs of SEQ ID NOs:8-10 and14-16, and CDRs specified by any of the following VH amino acidsequences: SEQ ID NOs: 6, 34-46, 88-91 and 93, including IGHV3-07,IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69, or IGHV3-48, which may comprise93, 92, 92, 99, 97, 97, 96, 96, 94, 94, 93, 92% identity respectively tothe FW region of DP-54 and one or fewer amino acid differences in commonstructural features (Kabat Numbering) (A) residues directly underneathCDR (Vernier Zone), H2, H47, H48, and H49, H67, H69, H71, H73, H93, H94,(B) VH/VL Chain packing Residues: H37, H39, H45, H47, H91, H93 and (C)canonical CDR Structural support residues H24, H71, H94 (see Lo 2004,and O'Brien and Jones 2003). Exemplary framework regions of DP-50,IGHV3-30*09, IGHV3-30*15 sharing 93, 92 and 92% identity to DP-54respectively and have no amino acid differences in these commonstructural features. In some aspects, the % identity is based onsimilarity with VH excluding those portions herein defined as CDRs.

In certain embodiments, the antibody, or antigen-binding fragmentthereof, described herein comprises (i) a VH comprising an amino acidsequence that is at least 50%, at least 60%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the amino acid sequence ofSEQ ID NO:6, and/or (ii) a VL comprising an amino acid sequence that isat least 50%, at least 60%, at least 66%, at least 70%, at least 75%, atleast 76%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to the aminoacid sequence of SEQ ID NO:7. Any combination of these VL and VHsequences is also encompassed by the invention.

In certain embodiments, the antibody, or antigen-binding fragmentthereof, described herein comprises (i) a HC comprising an amino acidsequence that is at least 50%, at least 60%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:3; and/or (ii) a LC comprising an amino acidsequence that is at least 50%, at least 60%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the amino acid sequence ofSEQ ID NO:5. Any combination of these HC and LC sequences is alsoencompassed by the invention.

In certain embodiments, the antibody, or antigen-binding fragmentthereof, described herein comprises an Fc domain. The Fc domain can bederived from IgA (e.g., IgA₁ or IgA₂), IgG, IgE, or IgG (e.g., IgG₁,IgG₂, IgG₃, or IgG₄).

The invention further provides an antibody, or antigen-binding fragmentthereof, that competes for binding to human αvβ8 integrin with LatencyAssociated Peptide (LAP). For example, if the binding of an antibody, orantigen-binding fragment thereof, to human αvβ8 integrin hinders thesubsequent binding of LAP to the human αvβ8 integrin, then the antibody,or the antigen-binding fragment thereof, competes with LAP for humanαvβ8 integrin binding.

The antibodies and antigen-binding fragments provided by the inventioninclude monoclonal antibodies, polyclonal antibodies, antibody fragments(e.g., Fab, Fab′, F(ab′)₂, Fv, Fc, etc.), chimeric antibodies,bispecific antibodies, heteroconjugate antibodies, single chain (ScFv),mutants thereof, fusion proteins comprising an antibody fragment, domainantibodies (dAbs), humanized antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site of the required specificity, including glycosylationvariants of antibodies, amino acid sequence variants of antibodies, andcovalently modified antibodies. The antibodies and antigen-bindingfragments may be murine, rat, human, or any other origin (includingchimeric or humanized antibodies). In some embodiments, the antibody isa monoclonal antibody. In some embodiments, the antibody is a chimeric,humanized or human antibody. In certain embodiments, the antibody is ahuman antibody. In certain embodiments, the antibody is a humanizedantibody.

Biological Activity of Anti-αvβ8 Integrin Antibodies

In addition to binding an epitope on αvβ8 integrin, the antibody, orantigen-binding fragment thereof, of the invention can mediate abiological activity. That is, the invention includes an isolatedantibody, or antigen-binding fragment thereof, that specifically bindsαvβ8 integrin and mediates at least one detectable activity selectedfrom the following:

(i) binds specifically to αvβ8 integrin (e.g., αvβ8 integrin from human,mouse, cynomolgus monkey, and/or rat);

(ii) reduces an interaction between αvβ8 integrin and Latency AssociatedPeptide (LAP);

(iii) reduces TGF-β signaling;

(iv) effectively blocks the αvβ8 integrin-mediated TGFβ activation withan IC50≤10 nM;

(v) has a comparable Kd (within 5-fold) towards a non-human primate(NHP) orthologue;

(vi) selectivity binds human αvβ8 and does not detectably bind ahomologue of αvβ8 (e.g., αvβ1, αvβ3, αvβ5 and αvβ6);

(vii) causes growth suppression and/or complete tumor regression in ananimal model for a cancer chosen from squamous cell carcinoma, breast,and colon cancer, alone or in combination with an immunomodulatory,e.g., a modulators of checkpoint inhibitors, e.g., inhibitors of PD-1,PD-L1, CTLA-4, or an agonist of a stimulatory molecule, e.g., 4-1BB;

(viii) causes growth suppression and/or complete tumor regression in ananimal model for a cancer in combination with an anti-cancer therapy,e.g., radiotherapy;

(ix) shows at least 60% reduction in tumor growth in a syngeneic tumorgraft model, e.g., when administered at ≤10 mg/kg;

(x) increases an anti-tumor response in the presence of one or moreimmunomodulators, e.g., an antagonist of a checkpoint inhibitor or anagonist of a checkpoint activator, e.g., an antagonist of PD-1, PD-L1,or CTLA-4, or an activator of an immune response, e.g., 4-1BB agonist,when administered to a subject, e.g., a mouse or human subject;

(xi) has an efficacy that is not dependent upon the expression of αvβ8integrin in a tumor model;

(xii) increases the abundance of CD8+ GzmB+ T cell in the tumormicroenvironment, e.g., as a monotherapy;

(xiii) shows a decrease, e.g., at least a >80% decrease, in tumor growthwhen used in combination with an antagonist of a checkpoint inhibitor(e.g., an anti-PD-1 or anti-PD-L1 antibody), e.g., in a syngeneic modelof squamous cell carcinoma, breast cancer, and/or colon cancer;

(xiv) shows a statistically significant improvement in overall survivalof a subject, e.g., a human or a mouse, as determined by a Kaplan-Meieranalysis;

(xv) shows suitable formulation properties, including a high degree ofthermal stability and minimal aggregation at high concentration; or

(xvi) may show reproducible expression and purity in large-scalemanufacturing conditions.

In some embodiments, the anti-αvβ8 integrin antibodies, orantigen-binding fragments thereof, has at least one of the followingproperties:

i. a binding affinity expressed as KD for human αvβ8 integrin that isless than the KD of the murine antibody ADWA11, e.g., less than 536 pM(e.g., 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250,300, 350, 370, 400, 450, 500, 510, 520, 530, 531, 532, 533, 534, or 535pM);

ii. a KD for human αvβ8 integrin that is less than or equal to 100 pM(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, or 100 pM), e.g., for purified human αvβ8 integrin;

iii. a KD for mouse αvβ8 integrin that is less than the KD of the murineantibody ADWA11, e.g., less than 489 pM (e.g., 1, 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 370, 400, 450, 460, 470,480, 485, 486, 487, or 488 pM);

iv. a KD for cynomolgus monkey αvβ8 integrin that is less than the KD ofthe murine antibody ADWA11, e.g., less than 507 pM (e.g., 1, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 370, 400, 450,500, 501, 502, 503, 504, 505, or 506 pM);

v. a KD for rat αvβ8 integrin that is about 160 pM;

vi. shows approximately equivalent affinity for at least two, three, orall of human, cynomolgus, mouse, and rat αvβ8 integrin, e.g., with a KDthat is less than 100 pM (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,30, 40, 50, 60, 70, 80, 90 or 95 pM), e.g., as determined using aBiacore affinity assay;

vii. an IC50 for inhibiting TGFβ transactivation that is less than themurine antibody ADWA11, e.g., less than 183 pM (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 175, 180, 181, or 182 pM);

viii. an IC50 for inhibiting TGFβ transactivation in U251 cells of about100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320 or 340 pM;

ix. an EC50 for U251 cells of about 126 pM with a standard deviation ofplus or minus 34 pM;

x. an EC50 for U251 cells of about 256 pM with a standard deviation ofplus or minus 115 pM;

xi. an EC50 for C8-S cells of about 115 pM;

xii. an EC50 for C8-S cells of about 145 pM with a standard deviation ofplus or minus 23.7 pM;

xiii. at least one predicted human pharmacokinetic (PK) parameter chosenfrom:

-   -   a. a clearance from central compartment (CL) of about 0.12-0.15        mL/h/kg;    -   b. an inter-compartmental distribution clearance (CLF) of about        0.15-0.51 mL/h/kg;    -   c. a volume of distribution for the central compartment (V1) of        about 36-39 mL/kg;    -   d. a volume of distribution for the peripheral compartment (V2)        of about 21-33 mL/kg; and/or    -   e. a terminal half-life (t_(1/2)) of about 12-17 days; or

xiv. shows no detectable binding to human Fcγ receptors or C1q.

In some embodiments, the anti-αvβ8 integrin antibodies, orantigen-binding fragments thereof, binds αvβ8 integrin+cells (e.g.,cells expressing αvβ8 integrin) with high apparent affinity. Apparentaffinity binding can be assessed using flow cytometry to detect antibodybinding to cells expressing the target protein (e.g., αvβ8 integrin).The cells can be transiently or stably transfected with a nucleic acidencoding αvβ8 integrin. Alternatively, the cells can be cells thatnaturally express αvβ8 integrin on their surface. Regardless of thesources of αvβ8 integrin+cells, the binding of the antibody to the cellscan be readily assessed using a variety of art-recognized methods. Theantibody, or antigen-binding fragment thereof, bind human αvβ8 integrin,cynomolgus monkey αvβ8 integrin, mouse αvβ8 integrin, rat αvβ8 integrin.

The invention includes an antibody, or antigen-binding fragment thereof,that specifically binds αvβ8 integrin and antagonizes an activitymediated by αvβ8 integrin (e.g., TGFβ signaling, which can be mediatedby inhibiting the interaction of αvβ8 integrin with the LatencyAssociated Peptide (LAP)). There are many assays known in the art todetermine the inhibition of an activity mediated by TGFβ signaling. Onesuch assay is a TGFβ pathway trans-activation assay. In one example ofsuch an assay, the expression of SMAD, which can serve as a marker ofTGFβ signaling and pathway activation, in monitored via the use of aluciferase reporter. The ability of the anti-αvβ8 integrin antibody tobind αvβ8 integrin and antagonize the effect of TGFβ signaling (e.g., byinhibiting the interaction of αvβ8 integrin with LAP) is thereforeassessed by measuring the level of SMAD expressed in the presence orabsence of the antibody. Preferably, the antibody can mediate adose-dependent decrease in luciferase with an EC50 of about 1 pM, about2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about8 pM, about 9 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM,about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about100 pM, about 125 pM, about 150 pM, about 175 pM, about 200, pM, about225 pM, about 250 pM, about 275 pM, about 300 pM, about 400 pM, or about500 pM (e.g., an EC50 of between about 1 pM and about 100 pM, e.g., anEC50 of between about 1 pM and about 200 pM, e.g., an EC50 of betweenabout 1 pM and about 300 pM, e.g., an EC50 of between about 1 pM andabout 400 pM, e.g., an EC50 of between about 1 pM and about 500 pM).More preferably, the antibody, or antigen-binding fragment thereof,inhibits TGFβ signaling (e.g., TGFβ transactivation, e.g., TGFβtransactivation of SMAD) with an EC50 of about 100 pM (e.g., an EC50 ofbetween about 5 pM and about 175 pM, e.g., an EC50 of between about 25pM and about 175 pM, e.g., an EC50 of about 100 pM, about 105 pM, about110 pM, about 115 pM, about 120 pM, about 125 pM, about 130 pM, about135 pM, about 140 pM, about 145 pM, about 150 pM, about 155 pM, about160 pM, about 165 pM, about 170 pM, or about 175 pM). In someembodiments, the antibody, or antigen-binding fragment thereof, inhibitsTGFβ signaling (e.g., TGFβ transactivation, e.g., TGFβ transactivationof SMAD) with an EC50 of less than about 5 nM (e.g., less than about0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.1, 5.5, 6, 7, 8, 9, 10, 15, 20, or 25 nM). Inembodiments, the antibody, or antigen-binding fragment thereof, inhibitsTGFβ signaling (e.g., TGFβ transactivation, e.g., TGFβ transactivationof SMAD) with an EC50 of about 5 nM (e.g., about 0.001, 0.01, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.1, 5.5, 6, 7, 8, 9, 10, 15, 20, or 25 nM).

In some embodiments, an antibody, or antigen-binding fragment thereof,of the present disclosure inhibits TGFβ signaling with an EC50 of about145+/−23.7 pM. In some embodiments, an antibody, or antigen-bindingfragment thereof, of the present disclosure inhibits TGFβ signaling inC8-S with an EC50 of about 145+/−23.7 pM. In some embodiments, anantibody or antigen-binding fragment thereof, of the present disclosureinhibits TGFβ signaling in C8-S cells with an EC50 of about 110 pM toabout 180 pM.

In some embodiments, an antibody, or antigen-binding fragment thereof,of the present disclosure inhibits TGFβ signaling with an EC50 of about256+/−115 pM. In some embodiments, an antibody, or antigen-bindingfragment thereof, of the present disclosure inhibits TGFβ signaling inU251 cells with an EC50 of about 256+/−115 pM. In some embodiments, anantibody or antigen-binding fragment thereof, of the present disclosureinhibits TGFβ signaling in U251 cells with an EC50 of about 80 pM toabout 400 pM.

The invention encompasses an antibody, or antigen-binding fragmentthereof, that binds human αvβ8 integrin but does not detectably bindhuman proteins αvβ3 integrin, αvβ5 integrin or αvβ6 integrin.

III. ANTI-αVβ8 INTEGRIN ANTIBODY EXPRESSION AND PRODUCTION NUCLEIC ACIDSENCODING ANTI-αVβ8 INTEGRIN ANTIBODIES

The invention also provides polynucleotides encoding any of theantibodies, including antibody fragments and modified antibodiesdescribed herein. The invention also provides a method of making any ofthe polynucleotides described herein. Polynucleotides can be made andexpressed by procedures known in the art.

The sequence of a desired antibody, defined antibody fragment, orantigen-binding fragment thereof, and nucleic acid encoding suchantibody, or fragment thereof, can be determined using standardsequencing techniques. A nucleic acid sequence encoding a desiredantibody, defined antibody fragment, or antigen-binding fragmentthereof, may be inserted into various vectors (such as cloning andexpression vectors) for recombinant production and characterization. Anucleic acid encoding the heavy chain, defined antibody fragment, orantigen-binding fragment of the heavy chain, and a nucleic acid encodingthe light chain, defined antibody fragment, or antigen-binding fragmentof the light chain, can be cloned into the same vector, or differentvectors.

The polynucleotide encoding the amino acid sequences above, encodes anamino acid sequence at least 80%. 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%, and more preferably identical to, the amino acidsequence of the antibodies, or antigen-binding fragment thereof, of thepresent invention as disclosed herein.

In some embodiments, the invention provides polynucleotides encoding oneor more proteins comprising the amino acid sequence selected from thegroup consisting of: SEQ ID NOs:2, 3, and 5-76 (e.g., a polynucleotidecomprising a sequence set forth in SEQ ID NO: 1, 4, 183, 185, 186, 189,190 or 191), or a nucleotide sequence encoding an amino acid sequence atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, andmore preferably identical to SEQ ID NOs:2, 3, 5-76, 88-93, 123, 124 and182.

The invention provides polynucleotides comprising the nucleic acidsequence as set forth as one or more of SEQ ID NOs: 1, 183, 189 or 191and encoding an antibody heavy chain or a nucleotide sequence at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, oridentical to SEQ ID NOs: 1, 183, 189 or 191 and encoding an antibodyheavy chain.

The invention provides polynucleotides comprising the nucleic acidsequence as set forth as one or more of SEQ ID NOs: 4 or 185 andencoding an antibody light chain or a nucleotide sequence at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or identicalto SEQ ID NOs: 4 or 185 and encoding an antibody light chain.

The invention provides polynucleotides comprising the nucleic acidsequence as set forth in SEQ ID NO: 190 and encoding an antibody heavychain variable region, or a nucleotide sequence at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or identical to SEQ IDNO: 190 and encoding an antibody heavy chain variable region.

The invention provides polynucleotides comprising the nucleic acidsequence as set forth in SEQ ID NO: 186 and encoding an antibody lightchain variable region, or a nucleotide sequence at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or identical to SEQ IDNO: 186 and encoding an antibody light chain variable region.

The invention provides polynucleotides comprising the nucleic acidsequence as set forth in SEQ ID NO: 192 or 193 and encoding an antibodyheavy chain constant region, or a nucleotide sequence at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or identical to SEQID NO: 192 or 193 and encoding an antibody heavy chain constant region.

The invention provides polynucleotides comprising the nucleic acidsequence as set forth in SEQ ID NO: 194 and encoding an antibody lightchain constant region, or a nucleotide sequence at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or identical to SEQ IDNO: 194 and encoding an antibody light chain constant region.

The invention provides a polynucleotide comprising the nucleic acidsequence as set forth as SEQ ID NO: 189. The invention provides apolynucleotide comprising the nucleic acid sequence as set forth as SEQID NO: 190. The invention provides a polynucleotide comprising thenucleic acid sequence as set forth as SEQ ID NO: 185.

The invention provides a polynucleotide comprising one or both of thenucleic acid sequence of the DNA insert of the plasmid deposited withthe ATCC and having Accession No. PTA-124917, and/or Accession No.PTA-124918.

The invention provides a polynucleotide comprising the nucleic acidsequence of the insert in the plasmid deposited with the ATCC and havingAccession No. PTA-124917, and/or Accession No. PTA-124918.

The invention provides cells comprising one or more nucleic acidmolecules as set forth in SEQ ID NOs: 1, 183 and 4.

The invention provides cells comprising one or more nucleic acidmolecules as set forth in SEQ ID NOs: 185, 189 and 190.

In another aspect, the invention provides polynucleotides and variantsthereof encoding an anti-αvβ8 integrin antibody (e.g., an anti-humanαvβ8 integrin antibody), wherein such variant polynucleotides share atleast 70%, at least 75%, at least 80%, at least 85%, at least 87%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to any of the specific nucleic acidsequences disclosed herein. These amounts are not meant to be limitingand increments between the recited percentages are specificallyenvisioned as part of the disclosure.

The invention provides polypeptides encoded by the nucleic acidmolecules described herein.

In one embodiment, the VH and VL domains, or antigen-binding fragmentthereof, or full length HC or LC, are encoded by separatepolynucleotides. Alternatively, both VH and VL, or antigen-bindingfragment thereof, or HC and LC, are encoded by a single polynucleotide.

Polynucleotides complementary to any such sequences are also encompassedby the present disclosure. Polynucleotides may be single-stranded(coding or antisense) or double-stranded, and may be DNA (genomic, cDNAor synthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present disclosure, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a fragment thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. In some embodiments, variants exhibit at least about 70%identity, in some embodiments, at least about 80% identity, in someembodiments, at least about 90% identity, and in some embodiments, atleast about 95% identity to a polynucleotide sequence that encodes anative antibody or a fragment thereof. These amounts are not meant to belimiting and increments between the recited percentages are specificallyenvisioned as part of the disclosure.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegAlign® program in the Lasergene® suite of bioinformatics software(DNASTAR®, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

In some embodiments, the “percentage of sequence identity” is determinedby comparing two optimally aligned sequences over a window of comparisonof at least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity. Variants may also, or alternatively, be substantiallyhomologous to a native gene, or a portion or complement thereof. Suchpolynucleotide variants are capable of hybridizing under moderatelystringent conditions to a naturally occurring DNA sequence encoding anative antibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/mL), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present disclosure.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present disclosure. Allelesare endogenous genes that are altered as a result of one or moremutations or alterations, such as deletions, additions and/orsubstitutions of nucleotides. The resulting mRNA and protein may, butneed not, have an altered structure or function. Alleles may beidentified using standard techniques (such as hybridization,amplification and/or database sequence comparison).

The polynucleotides of this disclosure can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, for example.

In some embodiments, a first vector comprises a polynucleotide thatencodes a heavy chain and a second vector comprises a polynucleotidethat encodes a light chain. In some embodiments, the first vector andsecond vector are transfected into host cells in similar amounts (suchas similar molar amounts or similar mass amounts). In some embodiments,a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and thesecond vector is transfected into host cells. In some embodiments, amass ratio of between 1:1 and 1:5 for the vector encoding the heavychain and the vector encoding the light chain is used. In someembodiments, a mass ratio of 1:2 for the vector encoding the heavy chainand the vector encoding the light chain is used.

Vectors

In some embodiments, a vector is selected that is optimized forexpression of polypeptides in CHO or CHO-derived cells, or in NSO cells.Exemplary such vectors are described, e.g., in Running Deer et al.,Biotechnol. Prog. 20:880-889 (2004).

Suitable cloning and expression vectors can include a variety ofcomponents, such as promoter, enhancer, and other transcriptionalregulatory sequences. The vector may also be constructed to allow forsubsequent cloning of an antibody variable domain into differentvectors. Suitable cloning vectors may be constructed according tostandard techniques, or may be selected from a large number of cloningvectors available in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.Expression vectors are further provided. Expression vectors generallyare replicable polynucleotide constructs that contain a polynucleotideaccording to the disclosure. It is implied that an expression vectormust be replicable in the host cells either as episomes or as anintegral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, andexpression vector(s) disclosed in PCT Publication No. WO 87/04462.Vector components may generally include, but are not limited to, one ormore of the following: a signal sequence; an origin of replication; oneor more marker genes; suitable transcriptional controlling elements(such as promoters, enhancers and terminator). For expression (i.e.,translation), one or more translational controlling elements are alsousually required, such as ribosome binding sites, translation initiationsites, and stop codons.

The vectors containing the polynucleotides of interest and/or thepolynucleotides themselves, can be introduced into the host cell by anyof a number of appropriate means, including electroporation,transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (e.g., where the vector is aninfectious agent such as vaccinia virus). The choice of introducingvectors or polynucleotides will often depend on features of the hostcell.

Host Cells

The antibody, or antigen-binding fragment thereof, may be maderecombinantly using a suitable host cell. A nucleic acid encoding theantibody or antigen-binding fragment thereof can be cloned into anexpression vector, which can then be introduced into a host cell, suchas E. coli cell, a yeast cell, an insect cell, a simian COS cell, aChinese hamster ovary (CHO) cell, or a myeloma cell where the cell doesnot otherwise produce an immunoglobulin protein, to obtain the synthesisof an antibody in the recombinant host cell. Preferred host cellsinclude a CHO cell, a human embryonic kidney HEK-293 cell, or an Sp2.0cell, among many cells well-known in the art. An antibody fragment canbe produced by proteolytic or other degradation of a full-lengthantibody, by recombinant methods, or by chemical synthesis. Apolypeptide fragment of an antibody, especially shorter polypeptides upto about 50 amino acids, can be conveniently made by chemical synthesis.Methods of chemical synthesis for proteins and peptides are known in theart and are commercially available.

In various embodiments, anti-αvβ8 integrin heavy chains and/or anti-αvβ8integrin light chains may be expressed in prokaryotic cells, such asbacterial cells; or in eukaryotic cells, such as fungal cells (such asyeast), plant cells, insect cells, and mammalian cells. Such expressionmay be carried out, for example, according to procedures known in theart. Exemplary eukaryotic cells that may be used to express polypeptidesinclude, but are not limited to, COS cells, including COS 7 cells; 293cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells.In some embodiments, anti-αvβ8 integrin heavy chains and/or anti-αvβ8integrin light chains may be expressed in yeast. See, e.g., U.S.Publication No. US 2006/0270045 A1. In some embodiments, a particulareukaryotic host cell is selected based on its ability to make desiredpost-translational modifications to the anti-αvβ8 integrin heavy chainsand/or anti-αvβ8 integrin light chains. For example, in someembodiments, CHO cells produce polypeptides that have a higher level ofsialylation than the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids into a desired host cell maybe accomplished by any method, including but not limited to, calciumphosphate transfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,etc. Nonlimiting exemplary methods are described, e.g., in Sambrook etal., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring HarborLaboratory Press (2001). Nucleic acids may be transiently or stablytransfected in the desired host cells, according to any suitable method.

Anti-αvβ8 integrin antibodies may be purified by any suitable method.Such methods include, but are not limited to, the use of affinitymatrices or hydrophobic interaction chromatography. Suitable affinityligands include ligands that bind antibody constant regions. Forexample, a Protein A, Protein G, Protein A/G, or an antibody affinitycolumn may be used to bind the constant region and to purify ananti-αvβ8 integrin antibody. Hydrophobic interactive chromatography, forexample, a butyl or phenyl column, may also suitable for purifying somepolypeptides. Many methods of purifying polypeptides are known in theart.

In some embodiments, an anti-αvβ8 integrin antibody is produced in acell-free system. Non-limiting exemplary cell-free systems aredescribed, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44(2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al.,Biotechnol. Adv. 21: 695-713 (2003).

IV. USES AND MEDICAL THERAPIES Therapeutic Uses

In some aspects, the invention provides for therapeutic methods forinhibiting αvβ8 integrin activity by using an anti-αvβ8 integrinantibody or antigen-binding fragment thereof, wherein the therapeuticmethods comprise administering a therapeutically effective amount of apharmaceutical composition comprising an anti-αvβ8 integrin antibody orantigen-binding fragment thereof. The disorder treated is any disease orcondition which is improved, ameliorated, inhibited, or prevented byremoval, inhibition, or reduction of αvβ8 integrin activity orsignaling. In particular, the anti-αvβ8 integrin antibodies of theinvention, including humanized and chimeric antibodies, can be used inthe prevention, treatment, and/or amelioration of diseases, disorders,or conditions caused by and/or associated with aberrant (e.g.,increased) αvβ8 integrin activity and/or TGFβ signaling in a subject(e.g., within the tumor microenvironment of a subject having a cancer).In some embodiments, the disease, disorder, or condition comprises arespiratory condition (e.g., asthma), fibrosis, or anemia. In someembodiments, the disease, disorder, or condition is treatable orpreventable with a vaccine.

In some aspects, the invention provides a method for the selectiveinhibition of αvβ8-dependent latent-TGFβ activation in cells in thetumor microenvironment including, for example, dendritic cells, Tregulatory cells, tumor associated macrophages, and/or the cells of thetumor itself. Without being bound by any particular theory, a moreprecise and selective antagonism of TGFβ activation within the immunesystem and/or the tumor microenvironment mediated by administration ofan anti-αvβ8 integrin antibody of the invention may contribute to ananti-tumor immune response in a subject without perturbing the broaderhomeostatic functions of TGFβ. Such an anti-tumor immune response withina subject that does not perturb the broader homeostatic functions ofTGFβ may be expected to provide safety and therapeutic advantages oversystemic TGFβ inhibition.

The methods provided herein may also be used to reduced and/or attenuateTGFβ activity, (e.g., TGFβ tumor-promoting activity) in a subject (e.g.,within a tumor microenvironment of a subject having a cancer). TGFβactivities influencing angiogenesis, metastasis, epithelial-mesenchymaltransition, and/or suppression of infiltrating immune cells (e.g., tumorinfiltrating lymphocytes, e.g., T cells, B cells, natural killer cells,macrophages, neutrophils, dendritic cells, mast cells, eosinophils, andbasophils) within a tumor microenvironment may be reduced and/orattenuated by the administration of a therapeutically effective amountof an anti-αvβ8 integrin antibody of the invention to a subject (e.g., asubject having a cancer).

In some instances, administration of an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, to a subject having a cancer increasesthe amount (e.g., density as determined by an immunohistochemistry (IHC)analysis) of infiltrating immune cells, for example, CD45 totallymphocytes and myeloid cells, CD3 T cells, CD4 T cells, CD8 T cells,and GranzymeB expressing cells (e.g., activated CD8 and NK cells), in atumor sample (e.g., a solid tumor sample) acquired from the subject. Anincrease in the amount (e.g., density) of infiltrating immune cells maybe determined, for example, by immunohistochemistry (IHC) analysis of atumor sample (e.g., a solid tumor sample) acquired from a subject havinga cancer in comparison to the amount (e.g., density) of infiltratingimmune cells acquired from a reference tumor sample (e.g., a tumorsample obtained from the same subject or a different subject having asimilar cancer, wherein the tumor sample was acquired prior to anadministration (e.g., a first administration, or a subsequentadministration) of an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof).

In one aspect, the invention relates to treatment of a subject in vivousing an anti-αvβ8 integrin antibody, or antigen-binding fragmentthereof, such that growth of cancerous tumors is inhibited or reduced.In some embodiments, a subject treated in vivo using an anti-αvβ8integrin antibody, or antigen-binding fragment thereof, has a primarycancer, for example a locally advanced cancer. In some embodiments, asubject treated in vivo using an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, has a recurrent cancer, for example ametastatic cancer.

In one aspect, the invention relates to in vivo treatment of a subjecthaving a cancer, or tumor, that expresses αvβ8 integrin, integrin (38(ITGβ8) and/or integrin αV (ITGαV) using an anti-αvβ8 integrin antibody,or antigen-binding fragment thereof. In some embodiments, expression ofαvβ8, β8 integrin and/or αV integrin is protein expression. In someembodiments, expression of αvβ8, β8 integrin and/or αV integrin is mRNAexpression. In some embodiments, expression of αvβ8, β8 integrin and/orαV integrin is increased expression relative to a level of αvβ8, β8integrin and/or αV integrin expression in a normal tissue or sample, ina control tissue or sample, in a tissue or sample prior to treatmentand/or a tissue or sample following treatment. In one embodiment, atissue or sample used to determine relative expression levels of αvβ8,β8 integrin and/or αV integrin may be obtained from a subject with acancer or tumor, from a different subject having the same type of canceror tumor or a different type of cancer or tumor or from a subjectwithout a cancer or tumor.

In some embodiments, mRNA expression of αvβ8, β8 integrin and/or αVintegrin is increased expression relative to a level of expression in anormal tissue or sample, in a control tissue or sample, in a tissue orsample prior to treatment and/or a tissue or sample following treatment.In some embodiments, mRNA expression of αvβ8, β8 integrin and/or αVintegrin is increased by about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about100%, about 150%, about 200%, about 300%, about 400%, about 500%relative to a level of mRNA expression of αvβ8, β8 integrin and/or αVintegrin in a normal tissue or sample, in a control tissue or sample, ina tissue or sample prior to treatment and/or a tissue or samplefollowing treatment.

An anti-αvβ8 integrin antibody, or antigen-binding fragment thereof, maybe used alone (e.g., as a monotherapy) to inhibit the growth of acancerous tumor. Alternatively, an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, may be used in combination with one ormore of: a standard of care treatment (e.g., for cancers or infectiousdisorders), another antibody or antigen-binding fragment thereof, animmunomodulator (e.g., an activator of a costimulatory molecule or aninhibitor of an inhibitory molecule); a vaccine, e.g., a therapeuticcancer vaccine; or other forms of cellular immunotherapy.

Accordingly, in one embodiment, the invention provides a method ofinhibiting growth of tumor cells in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-αvβ8integrin antibody, or antigen-binding fragment thereof, describedherein.

In one embodiment, the methods are suitable for the treatment of cancerin vivo. To achieve enhancement of immunity, the anti-αvβ8 integrinantibody, or antigen-binding fragment thereof, can be administeredtogether with an antigen of interest. When the anti-αvβ8 integrinantibody, or antigen-binding fragment thereof, is administered incombination with one or more agents, the combination can be administeredin either order or simultaneously.

In another aspect, a method of treating a subject, e.g., reducing orameliorating, a hyperproliferative condition or disorder (e.g., acancer), e.g., solid tumor, a hematological cancer, soft tissue tumor,or a metastatic lesion, in a subject is provided. The method includesadministering to the subject one or more anti-αvβ8 integrin antibodies,or antigen-binding fragments thereof, described herein, alone or incombination with other agents or therapeutic modalities. In someembodiments, the anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, of the invention can be administered as a 1^(st) linetherapy in treatment naïve subjects, or as a 2^(nd) line therapyfollowing for example, relapse or progression of the cancer.

Exemplary cancers whose growth can be treated, e.g., reduced, using theantibodies molecules disclosed herein include cancers typicallyresponsive to immunotherapy. Non-limiting examples of cancers fortreatment include melanoma (e.g., metastatic malignant melanoma, skincutaneous melanoma), renal cell cancer (RCC) (e.g., clear cellcarcinoma, papillary cell carcinoma), prostate cancer (e.g., hormonerefractory prostate adenocarcinoma), breast cancer, ovarian cancer(e.g., epithelial ovarian cancer, fallopian tube or primary peritonealcancer), head and neck cancer (e.g., squamous cell carcinoma of the headand neck (SCCHN)), colon cancer, esophageal cancer (e.g., adenocarcinomaand squamous cell carcinoma), gastric cancer (e.g., adenocarcinoma ofgastric and gastroesophageal junction), pancreatic cancer (e.g,pancreatic ductal adenocarcinoma), biliary duct cancer (e.g.,cholangiocarcinoma); endometrial cancer (e.g., uterine corpusendometrial carcinoma), urothelial carcinoma and lung cancer (e.g.,non-small cell lung cancer, squamous cell cancer). Additionally,refractory or recurrent malignancies can be treated using the antibodymolecules described herein.

Examples of other cancers that can be treated include bone cancer,cutaneous or intraocular malignant melanoma, rectal cancer, anal cancer,testicular cancer, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, Merkel cell cancer, Hodgkin lymphoma,non-Hodgkin lymphoma, cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, chronic or acute leukemias includingacute myeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, multiple myeloma,myelodysplastic syndromes, cancer of the kidney or ureter, carcinoma ofthe renal pelvis, neoplasm of the central nervous system (CNS), primaryCNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma,pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cellcancer, T-cell lymphoma, environmentally induced cancers including thoseinduced by asbestos (e.g., mesothelioma), and combinations of saidcancers. In some embodiments, the cancer is chosen from a renal cellcarcinoma, an ovarian cancer, a head and neck squamous cell carcinoma,and a skin cancer (e.g., a melanoma, e.g., an advanced melanoma).

In some instances, the cancer is selected from the group consisting of asolid tumor, a hematological cancer (e.g., leukemia, lymphoma, myeloma,e.g., multiple myeloma), and a metastatic lesion. The cancer may be asolid tumor, for example, a solid tumor such as a malignancies, e.g.,sarcomas and carcinomas, e.g., adenocarcinomas of the various organsystems, such as those affecting the lung (e.g., a non-small cell lungcancer (NSCLC)), breast, ovarian, lymphoid, gastrointestinal (e.g.,colon), anal, genitals and genitourinary tract (e.g., renal, urothelial,bladder cells, prostate), pharynx, CNS (e.g., brain, neural or glialcells), head and neck (e.g., head and neck squamous cell carcinoma(HNSCC), skin (e.g., melanoma, e.g., an advanced melanoma), pancreas,colon, rectal, a renal (e.g., a renal cell carcinoma), liver, cancer ofthe small intestine and cancer of the esophagus, gastro-esophagealcancer, thyroid cancer, and cervical cancer. In some instances, thecancer may be a lymphoproliferative disease (e.g., a post-transplantlymphoproliferative disease) or a hematological cancer, T-cell lymphoma,B-cell lymphoma, a non-Hodgkin lymphoma, or a leukemia (e.g., a myeloidleukemia or a lymphoid leukemia). In some instances, the cancer is anearly, intermediate, late stage or metastatic cancer. In particularinstances, the cancer is a renal cell carcinoma, an ovarian cancer, or ahead and neck squamous cell carcinoma.

In other embodiments, the cancer is a hematological malignancy or cancerincluding but is not limited to a leukemia or a lymphoma. For example,the anti-αvβ8 integrin antibody, or antigen-binding fragment thereof,can be used to treat cancers and malignancies including, but not limitedto, e.g., acute leukemias including but not limited to, e.g., B-cellacute lymphoid leukemia (“BALL”), T-cell acute lymphoid leukemia(“TALL”), acute lymphoid leukemia (ALL); one or more chronic leukemiasincluding but not limited to, e.g., chronic myelogenous leukemia (CML),chronic lymphocytic leukemia (CLL); additional hematologic cancers orhematologic conditions including, but not limited to, e.g., B cellprolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm,Burkitt's lymphoma, diffuse large B cell lymphoma, Follicular lymphoma,Hairy cell leukemia, small cell- or a large cell-follicular lymphoma,malignant lymphoproliferative conditions, MALT lymphoma, mantle celllymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and“preleukemia” which are a diverse collection of hematological conditionsunited by ineffective production (or dysplasia) of myeloid blood cells,and the like.

In one embodiment, the cancer is chosen from a lung cancer (e.g., anon-small cell lung cancer (NSCLC) (e.g., a NSCLC with squamous and/ornon-squamous histology, or a NSCLC adenocarcinoma)), a melanoma (e.g.,an advanced melanoma), a cutaneous squamos cell carcinoma (cutaneousSCC) (e.g., metastatic cutaneous SCC), a renal cancer (e.g., a renalcell carcinoma, e.g., clear cell renal cell carcinoma), a liver cancer,a myeloma (e.g., a multiple myeloma), a prostate cancer, a breast cancer(e.g., a breast cancer that does not express one, two or all of estrogenreceptor, progesterone receptor, or Her2/neu, e.g., a triple negativebreast cancer), a colorectal cancer, a pancreatic cancer, a head andneck cancer (e.g., head and neck squamous cell carcinoma (HNSCC), analcancer, gastro-esophageal cancer, thyroid cancer, cervical cancer, alymphoproliferative disease (e.g., a post-transplant lymphoproliferativedisease) or a hematological cancer, T-cell lymphoma, a non-Hodgkin'slymphoma, or a leukemia (e.g., a myeloid leukemia).

In another embodiment, the cancer is chosen from a carcinoma (e.g.,advanced or metastatic carcinoma), melanoma or a lung carcinoma, e.g., anon-small cell lung carcinoma.

In one embodiment, the cancer is a lung cancer, e.g., a non-small celllung cancer.

In another embodiment, the cancer is a prostate cancer, e.g., anadvanced prostate cancer.

In yet another embodiment, the cancer is a myeloma, e.g., multiplemyeloma.

In yet another embodiment, the cancer is a renal cancer, e.g., a renalcell carcinoma (RCC) (e.g., a metastatic RCC or clear cell renal cellcarcinoma).

In some embodiments, when the cancer is a skin cancer (e.g., a melanoma,e.g., an advanced melanoma, e.g., cutaneous squamos cell carcinoma,e.g., metastatic cutaneous squamos cell carcinoma).

In one embodiment, the cancer is a melanoma, e.g., an advanced melanoma.In one embodiment, the cancer is an advanced or unresectable melanomathat does not respond to other therapies.

In some embodiments, the cancer is a renal cancer, e.g., a renal cellcarcinoma (RCC). In some instances, the renal cancer is a metastaticRCC, a clear cell renal cell carcinoma (ccRCC)), or a non-clear-cellrenal cell carcinoma (ncRCC). In some instances, when the cancer is anRCC, e.g., ccRCC, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, of the invention may be administered as a 1^(st) lineor 2^(nd) line therapy.

In some embodiments, when the cancer is an ovarian cancer an anti-αvβ8integrin antibody, or antigen-binding fragment thereof, of the inventionmay be administered as a 2^(nd) Line therapy for platinum-resistantpatients.

In some embodiments, the cancer is platinum-resistant and/or recurrentcancer.

Methods and compositions disclosed herein are useful for treatingmetastatic lesions associated with the aforementioned cancers.

Combination Therapies

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-beta (Kehrl, J. et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard, M. & O'Garra, A. (1992) Immunology Today 13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Antibodies or antigen-binding fragments thereof to each ofthese entities may be used in combination with anti-αvβ8 integrinantibodies, or antigen-binding fragments thereof, to counteract theeffects of the immunosuppressive agent and favor tumor immune responsesby the host.

The antibodies or antigen binding fragments disclosed herein can be usedin unconjugated forms or conjugated to a second agent, e.g., a cytotoxicdrug, radioisotope, or a protein, e.g., a protein toxin or a viralprotein. This method includes: administering the antibody molecule,alone or conjugated to a cytotoxic drug, to a subject requiring suchtreatment. The antibody molecules can be used to deliver a variety oftherapeutic agents, e.g., a cytotoxic moiety, e.g., a therapeutic drug,a radioisotope, molecules of plant, fungal, or bacterial origin, orbiological proteins (e.g., protein toxins) or particles (e.g.,recombinant viral particles, e.g.; via a viral coat protein), ormixtures thereof.

In certain embodiments, an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, can be used in combination with othertherapies to provide an unexpected synergistic therapeutic effect thatcan provide an effect greater than a merely additive effect of addingtwo individual therapies. For example, the combination therapy caninclude a composition of the present invention co-formulated with,and/or co-administered with, one or more additional therapeutic agents,e.g., one or more anti-cancer agents, cytotoxic or cytostatic agents,hormone treatment, vaccines, and/or other immunotherapies. In otherembodiments, the antibody molecules are administered in combination withother therapeutic treatment modalities, including surgery, radiation,cryosurgery, and/or thermotherapy. Such synergistic combinationtherapies may advantageously utilize lower dosages of the administeredtherapeutic agents, thus αvoiding possible toxicities or complicationsassociated with the various monotherapies.

In certain embodiments, the methods and compositions described hereinare administered in combination with one or more of other antibodymolecules, chemotherapy, other anti-cancer therapy (e.g., targetedanti-cancer therapies, or oncolytic drugs), cytotoxic agents,immune-based therapies (e.g., cytokines), surgical and/or radiationprocedures. Exemplary cytotoxic agents that can be administered incombination with include antimicrotubule agents, topoisomeraseinhibitors, anti-metabolites, mitotic inhibitors, alkylating agents,anthracyclines, vinca alkaloids, intercalating agents, agents capable ofinterfering with a signal transduction pathway, agents that promoteapoptosis, proteosome inhibitors, and radiation (e.g., local or wholebody irradiation).

Alternatively, or in combination with the aforesaid combinations, themethods and compositions described herein can be administered incombination with one or more of: an immunomodulator (e.g., an activatorof a costimulatory molecule or an inhibitor of an inhibitory molecule);a vaccine, e.g., a therapeutic cancer vaccine; or other forms ofcellular immunotherapy.

Exemplary non-limiting synergistic combinations and uses of an anti-αvβ8integrin antibodies, or antigen-binding fragments thereof, include thefollowing.

In certain embodiments, an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, is administered in combination with amodulator of a costimulatory molecule or an inhibitory molecule, e.g., aco-inhibitory ligand or receptor.

In one embodiment, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, is administered in combination with a modulator, e.g.,agonist, of a costimulatory molecule. In one embodiment, the agonist ofthe costimulatory molecule is chosen from an agonist (e.g., an agonisticantibody or antigen-binding fragment thereof, or soluble fusion) of4-1BB (CD137), OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS(CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7,NKp80, CD160, or B7-H3.

In another embodiment, an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, is used in combination with animmunomodulator, e.g., a costimulatory molecule, e.g., an agonist ormodulator associated with a positive signal that includes acostimulatory domain of 4-1BB (CD137), CD28, CD27, ICOS and GITR.

Exemplary GITR modulators include, e.g., GITR fusion proteins andanti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as, aGITR fusion protein described in U.S. Pat. No. 6,111,090, EuropeanPatent No.: 090505B1, U.S. Pat. No. 8,586,023, PCT Publication Nos.: WO2010/003118 and 2011/090754, or an anti-GITR antibody described, e.g.,in U.S. Pat. No. 7,025,962, European Patent No.: 1947183B1, U.S. Pat.Nos. 7,812,135, 8,388,967, 8,591,886, European Patent No.: EP 1866339,PCT Publication No.: WO 2011/028683, PCT Publication No.: WO2013/039954, PCT Publication No.: WO2005/007190, PCT Publication No.: WO2007/133822, PCT Publication No.: WO2005/055808, PCT Publication No.: WO99/40196, PCT Publication No.: WO 2001/03720, PCT Publication No.:WO99/20758, PCT Publication No.: WO2006/083289, PCT Publication No.: WO2005/115451, U.S. Pat. No. 7,618,632, and PCT Publication No.: WO2011/051726. In one embodiment, an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, is administered in combination with aninhibitor of an inhibitory molecule of an immune checkpoint molecule. Itwill be understood by those of ordinary skill in the art, that the term“immune checkpoints” means a group of molecules on the cell surface ofCD4 and CD8 T cells. These molecules can effectively serve as “brakes”to down-modulate or inhibit an anti-tumor immune response. Immunecheckpoint molecules include, but are not limited to, Programmed Death 1(PD-1), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), B7H1, B7H4, OX-40,CD137, CD40, LAG-3 and TIM-3, which directly inhibit immune cells.Immunotherapeutic agents which can act as immune checkpoint inhibitorsuseful in the methods of the present invention, include, but are notlimited to, inhibitors of PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM, and/or TGF beta.Inhibition of an inhibitory molecule can be performed by inhibition atthe DNA, RNA or protein level. In embodiments, an inhibitory nucleicacid (e.g., a dsRNA, siRNA or shRNA), can be used to inhibit expressionof an inhibitory molecule. In other embodiments, the inhibitor of aninhibitory signal is, a polypeptide e.g., a soluble ligand, or anantibody or antigen-binding fragment thereof, that binds to theinhibitory molecule.

In one embodiment, the inhibitor is a soluble ligand (e.g., a CTLA-4-Igor a TIM-3-Ig), or an antibody or antibody fragment that binds to PD-1,PD-L1, PD-L2 or CTLA4. For example, an anti-PD-1 antibody molecule canbe administered in combination with an anti-CTLA-4 antibody, e.g.,ipilimumab, for example, to treat a cancer (e.g., a cancer chosen from:a melanoma, e.g., a metastatic melanoma; a lung cancer, e.g., anon-small cell lung carcinoma; or a prostate cancer). Exemplaryanti-CTLA4 antibodies include Tremelimumab (IgG2 monoclonal antibodyavailable from Pfizer, formerly known as ticilimumab, CP-675,206); andIpilimumab (CTLA-4 antibody, also known as MDX-010, CAS No.477202-00-9). In one embodiment, an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, is administered after treatment, e.g.,after treatment of a melanoma, with an anti-CTLA4 antibody (e.g.,ipilimumab) with or without a BRAF inhibitor (e.g., vemurafenib ordabrafenib). Exemplary doses of an anti-CTLA-4 antibody, e.g.,ipilimumab, of about 3 mg/kg.

Other antibodies which may be used to activate host immuneresponsiveness can be used in combination with an anti-αvβ8 integrinantibody, or antigen-binding fragment thereof. These include moleculeson the surface of dendritic cells which activate DC function and antigenpresentation. Anti-CD40 antibodies are able to substitute effectivelyfor T cell helper activity (Ridge, J. et al. (1998) Nature 393: 474-478)and can be used in conjunction with an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof. Antibodies to T cell costimulatorymolecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40(Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I.et al. (1997) Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff, A.et al. (1999) Nature 397: 262-266) may also provide for increased levelsof T cell activation.

In certain embodiments, an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, described herein is administered incombination with one or more inhibitors of PD-1, PD-L1 and/or PD-L2known in the art. In one embodiment, an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, is administered concurrently with animmune checkpoint inhibitor (e.g., an anti-PD-1, anti-PD-L1 and/oranti-PD-L2 antibody, or antigen-binding fragment thereof) to a subjectwho has not been previously treated with an immune checkpoint inhibitor.In one embodiment, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, is administered concurrently with an immune checkpointinhibitor (e.g., an anti-PD-1, anti-PD-L1 and/or anti-PD-L2 antibody, orantigen-binding fragment thereof) to a subject who has been previouslytreated with an immune checkpoint inhibitor and in whom the cancer ortumor had progressed (e.g., locally advanced, metastasized). In oneembodiment, an anti-αvβ8 integrin antibody, or antigen-binding fragmentthereof, is administered biweekly (e.g., every 2 week) in a 28-day cycleand an immune checkpoint inhibitor is administered every 4 weeks on day1 of each 28-day cycle. In one embodiment, an anti-αvβ8 integrinantibody, or antigen-binding fragment thereof, is administered biweeklyon a 28-day cycle and an immune checkpoint inhibitor (e.g., ananti-PD-1, anti-PD-L1 and/or anti-PD-L2 antibody, or antigen-bindingfragment thereof) is administered every 4 weeks on day 1 of each 28-daycycle to a subject who has a cancer or a tumor. In one embodiment, animmune checkpoint inhibitor is an anti-PD1 antibody, or antigen-bindingfragment thereof, described in PCT Publication No. WO2016/092419, (e.g.,RN888, as referred to as PF-06801591 or sasanlimab). In one embodiment,a cancer or tumor is squamous cell carcinoma, e.g., squamous cellcarcinoma of the head or neck (SCCHN), renal cell carcinoma (RCC),breast cancer, or colon cancer.

An inhibitor of PD-1, PD-L1 and/or PD-L2 may be an antibody, an antigenbinding fragment thereof, an immunoadhesin, a fusion protein, oroligopeptide. In some embodiments, an anti-PD-1 antibody is chosen fromMDX-1106, Merck 3475 or CT-011. In one embodiment, an anti-PD-1 antibodyis nivolumab/Opdivo®, pembrolizumab/Keytruda®, spartalizumab,pidilizumab, tislelizumab, AMP-224, AMP-514, cemiplimab, or sasanlimab(RN888, mAb7, PF-06801591). In one embodiment, an anti-PD-L1 antibody isMEDI4736, MPDL3280A (YW243.55.s70), BMS-936559 (MDX-1105),atezolizumab/Tecentriq®, durvalumab/Imfizi® or avelumab/Bavencio®. Inone embodiment, the anti-PD-L1 antibody is not avelumab. In oneembodiment, an anti-PD-1 antibody is described in PCT Publication No.WO2016/092419, including, but not limited to, mAb7 (also referred to asRN888, PF-06801591, or sasanlimab).

In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., animmunoadhesin comprising an extracellular or PD-1 binding portion ofPD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of animmunoglobulin sequence). In some embodiments, the PD-1 inhibitor isAMP-224. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1antibody. In some embodiments, an anti-PD-L1 binding antagonist ischosen from YW243.55.s70 (also known as MPDL3280A, atezolizumab),MEDI-4736, MSB-0010718C, or MDX-1105. MDX-1105, also known asBMS-936559, is an anti-PD-L1 antibody described in WO2007/005874.Antibody YW243.55.s70 also referred to as MPDL3280A (heavy and lightchain variable region sequences shown in SEQ ID Nos. 20 and 21,respectively) is an anti-PD-L1 described in WO 2010/077634.

MDX-1106, also known as MDX-1106-04, ONO-4538 or BMS-936558, is ananti-PD-1 antibody described in WO2006/121168. Merck 3745, also known asMK-3475 or SCH-900475, is an anti-PD-1 antibody described inWO2009/114335. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1kmonoclonal antibody that binds to PD-1. Pidilizumab and other humanizedanti-PD-1 monoclonal antibodies are disclosed in WO2009/101611. In otherembodiments, an anti-PD-1 antibody is pembrolizumab. Pembrolizumab(Trade name KEYTRUDA formerly lambrolizumab, also known as MK-3475)disclosed, e.g., in Hamid, O. et al. (2013) New England Journal ofMedicine 369 (2): 134-44. AMP-224 (B7-DCIg; Amplimmune; e.g., disclosedin WO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion solublereceptor that blocks the interaction between PD-1 and B7-H1. Otheranti-PD-1 antibodies include AMP-514 (Amplimmune), among others, e.g.,anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,609,089, US2010028330, and/or US 20120114649.

In some embodiments, an anti-PD-1 antibody is MDX-1106. Alternativenames for MDX-1106 include MDX-1106-04, ONO-4538, BMS-936558 ornivolumab. In some embodiments, an anti-PD-1 antibody is nivolumab (CASRegistry Number: 946414-94-4). Nivolumab (also referred to as BMS-936558or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonalantibody which specifically blocks PD-1. Nivolumab (clone 5C4) and otherhuman monoclonal antibodies that specifically bind to PD-1 are disclosedin U.S. Pat. No. 8,008,449 and WO2006/121168. Lambrolizumab (alsoreferred to as pembrolizumab or MK03475; Merck) is a humanized IgG4monoclonal antibody that binds to PD-1. Pembrolizumab and otherhumanized anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,354,509and WO2009/114335. MDPL3280A (Genentech/Roche) is a human Fc optimizedIgG1 monoclonal antibody that binds to PD-L1. MDPL3280A (also known asYW243.55.s70, and other human monoclonal antibodies to PD-L1 aredisclosed in U.S. Pat. No. 7,943,743 and U.S Publication No.20120039906. The sequences of YW243.55.s70 (heavy and light chainvariable regions are shown in SEQ ID NOs 20 and 21) are also set forthin WO2010/077634 and U.S. Pat. No. 8,217,149). MDX-1105 (also referredto as BMS-936559), and other anti-PD-L1 binding agents are disclosed inWO2007/005874.

In other embodiments, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, is administered in combination with a cytokine, e.g.,interleukin-21, interleukin-2, interleukin-12, or interleukin-15. Incertain embodiments, the combination of anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, and cytokine described herein is usedto treat a cancer, e.g., a cancer as described herein (e.g., a solidtumor or melanoma).

In all of the methods described herein, anti-αvβ8 integrin antibodies,or antigen-binding fragments thereof, can be combined with other formsof immunotherapy such as cytokine treatment (e.g., interferons, GM-CSF,G-CSF, IL-2, IL-21), or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens (see e.g., Holliger (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

Exemplary immunomodulators that can be used in combination with ananti-αvβ8 integrin antibody, or antigen-binding fragment thereof,include, but are not limited to, e.g., afutuzumab (available fromRoche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®);thalidomide (Thalomid®), actimid (CC4047); and cytokines, e.g., IL-21 orIRX-2 (mixture of human cytokines including interleukin 1, interleukin2, and interferon γ, CAS 951209-71-5, available from IRX Therapeutics).

In some embodiments, the cancer is ovarian cancer and an anti-αvβ8integrin antibody, or antigen-binding fragment thereof, is administeredin combination with an inhibitor of PARP1 (e.g., olaparib, rucaparib,niraparib, veliparib, iniparib, talazoparib, 3-aminobenzamide, CEP 9722,E7016, BSI-201, KU-0059436, AG014699, MK-4827, or BGB-290).

In some embodiments, the cancer is a head and neck cancer, e.g., a headand neck squamous cell carcinoma. In some embodiments, an anti-αvβ8integrin antibody, or antigen-binding fragment thereof, is administeredin combination with a radiation therapy.

Diagnostic Uses

Anti-αvβ8 integrin antibodies, or antigen-binding fragments thereof, ofthe present invention may also be used to detect and/or measure αvβ8integrin, or αvβ8 integrin-expressing cells in a sample, e.g., fordiagnostic purposes. For example, an anti-αvβ8 integrin antibody, orfragment thereof, may be used to diagnose a condition or diseasecharacterized by aberrant expression (e.g., over-expression,under-expression, lack of expression, etc.) of αvβ8 integrin. Exemplarydiagnostic assays for αvβ8 integrin may comprise, e.g., contacting asample, obtained from a patient, with an anti-anti-αvβ8 integrinantibody, or antigen-binding fragment thereof, of the invention, whereinan anti-αvβ8 integrin antibody, or antigen-binding fragment thereof, islabeled with a detectable label or reporter molecule.

In one aspect, the present invention provides a diagnostic method fordetecting the presence of an αvβ8 integrin protein in vitro (e.g., in abiological sample, such as a tissue biopsy, e.g., from a canceroustissue) or in vivo (e.g., in vivo imaging in a subject). The methodincludes: (i) contacting the sample with an antibody, or antigen-bindingfragment thereof, described herein, or administering to the subject, theantibody, or antigen-binding fragment thereof; (optionally) (ii)contacting a reference sample, e.g., a control sample (e.g., a controlbiological sample, such as plasma, tissue, biopsy) or a controlsubject)); and (iii) detecting formation of a complex between theantibody, or antigen-binding fragment thereof, and the sample orsubject, or the control sample or subject, wherein a change, e.g., astatistically significant change, in the formation of the complex in thesample or subject relative to the control sample or subject isindicative of the presence of αvβ8 integrin in the sample. The antibodymolecule can be directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound antibody.Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials, as described above and described in more detail below.

The term “sample,” as it refers to samples used for detectingpolypeptides includes, but is not limited to, cells, cell lysates,proteins or membrane extracts of cells, body fluids, or tissue samples.

Complex formation between the antibody molecule and αvβ8 integrin can bedetected by measuring or visualizing either the binding molecule boundto the αvβ8 integrin antigen or unbound binding molecule. Conventionaldetection assays can be used, e.g., an enzyme-linked immunosorbentassays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry.Alternative to labeling the antibody, or antigen-binding fragmentthereof, the presence of αvβ8 integrin can be assayed in a sample by acompetition immunoassay utilizing standards labeled with a detectablesubstance and an unlabeled antibody molecule. In this assay, thebiological sample, the labeled standards and the antibody, orantigen-binding fragment thereof, are combined and the amount of labeledstandard bound to the unlabeled binding molecule is determined. Theamount of αvβ8 integrin in the sample is inversely proportional to theamount of labeled standard bound to the antibody, or antigen-bindingfragment thereof.

V. COMPOSITIONS

The disclosure also provides pharmaceutical compositions comprising aneffective amount of an anti-αvβ8 integrin antibodies described herein.Examples of such compositions, as well as how to formulate, are alsodescribed herein. In some embodiments, the composition comprises one ormore anti-αvβ8 integrin antibodies. In some embodiments, an anti-αvβ8integrin antibody recognizes αvβ8 integrin (e.g., αvβ8 integrin from ahuman, mouse, cynomolgus monkey, or rat). In some embodiments, ananti-αvβ8 integrin antibody is a humanized antibody. In someembodiments, an anti-αvβ8 integrin antibody is a chimeric antibody. Insome embodiments, an anti-αvβ8 integrin antibody comprises a constantregion that is capable of triggering a desired immune response, such asantibody-mediated lysis or ADCC.

The composition used in the present disclosure can further comprisepharmaceutically acceptable carriers, excipients, or stabilizers, in theform of lyophilized formulations or aqueous solutions.

As used herein, “pharmaceutically acceptable carrier” or “pharmaceuticalacceptable excipient” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline (PBS) or normal (0.9%) saline. Compositions comprising suchcarriers are formulated by conventional methods (see, for example,Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., MackPublishing Co., Easton, Pa., 1990; and Remington, The Science andPractice of Pharmacy, 20th Ed., Mack Publishing, 2000).

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations, and may comprise bufferssuch as phosphate, citrate, and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutically acceptable excipients are further described herein.

Anti-αvβ8 integrin antibodies and compositions thereof can also be usedin conjunction with other agents, including an additional therapeuticagent (e.g., an inhibitor of an immune checkpoint molecule) that servesto enhance and/or complement the effectiveness of the agents.

The disclosure also provides compositions, including pharmaceuticalcompositions, comprising polynucleotides encoding antibodies of thedisclosure. In some embodiments, the composition comprises an expressionvector comprising a polynucleotide encoding an antibody as describedherein. In some embodiments, a composition comprises either or both ofthe polynucleotides of SEQ ID NOs: 1 or 4. In some embodiments, acomposition comprises either or both of the polynucleotides of SEQ IDNOs: 183 or 4. In some embodiments, a composition comprises either orboth of the polynucleotides of SEQ ID NOs: 185 or 189. In someembodiments, a composition comprises either or both of thepolynucleotides of SEQ ID NOs: 185 or 191.

In another aspect, the polynucleotide can encode the VH, VL and/or bothVH and VL of an antibody of the disclosure. That is, the compositioncomprises a single polynucleotide or more than one polynucleotideencoding the antibody, or antigen-binding fragment thereof, of thedisclosure.

The pharmaceutical compounds of the disclosure may include one or morepharmaceutically acceptable salts. Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the disclosure also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure suitable to high drug concentration. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In many cases, it will besuitable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration.

Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

A pharmaceutical composition of the present disclosure may be prepared,packaged, or sold in a formulation suitable for ophthalmicadministration. Such formulations may, for example, be in the form ofeye drops including, for example, a 0.1%-1.0% (w/w) solution orsuspension of the active ingredient in an aqueous or oily liquidcarrier. Such drops may further comprise buffering agents, salts, or oneor more other of the additional ingredients described herein. Otherophthalmically-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form or in aliposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the disclosure are knownin the art and described, for example in Remington's PharmaceuticalSciences, Genaro, ed., Mack Publishing Co., Easton, Pa. (1985), which isincorporated herein by reference.

In one embodiment, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, is formulated in a vial containing 100 mg of anti-αvβ8integrin antibody, or antigen-binding fragment thereof, in 1 mL ofaqueous buffered solution.

In one embodiment, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, is administered in an intravenous formulation as asterile aqueous solution containing about 0.1 mg/mL, about 1 mg/mL,about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, 5 mg/mL, or in someembodiments, about 10 mg/mL, or in some embodiments, about 15 mg/mL, orin some embodiments, about 20 mg/mL of antibody, or in some embodiments,about 25 mg/mL, or in some embodiments, about 50 mg/mL, or in someembodiments, about 100 mg/mL and 5% dextrose. In some embodiments, anintravenous formulation is a sterile aqueous solution containing 0.1mg/mL to 15 mg/mL of anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof in 5% dextrose. In some embodiments, an anti-αvβ8integrin antibody, or antigen-binding fragment thereof is formulated ina composition containing sodium acetate, polysorbate 80, and sodiumchloride at a pH ranging from about 5 to 6. In some embodiments, theintravenous formulation is a sterile aqueous solution containing 5 or 10mg/mL of antibody, with 20 mM sodium acetate, 0.2 mg/mL polysorbate 80,and 140 mM sodium chloride at pH 5.5. Further, a solution comprising anantibody, or antigen-binding fragment thereof, can comprise, among manyother compounds, histidine, mannitol, sucrose, trehalose, glycine,poly(ethylene) glycol, EDTA, methionine, and any combination thereof,and many other compounds known in the relevant art.

In one embodiment, a pharmaceutical composition of the presentdisclosure comprises the following components: 50 mg/mL anti-αvβ8integrin antibody or antigen-binding fragment of the present disclosure,20 mM histidine, 8.5% sucrose, and 0.02% polysorbate 80, 0.005% EDTA atpH 5.8; in another embodiment a pharmaceutical composition of thepresent invention comprises the following components: 100 mg/mLanti-αvβ8 integrin antibody or antigen-binding fragment of the presentdisclosure, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80 at pH5.8. This composition may be provided as a liquid formulation or as alyophilized powder. When the powder is reconstituted at full volume, thecomposition retains the same formulation. Alternatively, the powder maybe reconstituted at half volume, in which case the composition comprises100 mg anti-αvβ8 integrin antibody or antigen-binding fragment thereofof the present disclosure, 20 mM histidine, 10% sucrose, and 0.02%polysorbate 80 at pH 5.8.

In one embodiment, part of the dose is administered by an intravenousbolus and the rest by infusion of the antibody formulation. For example,a 0.01 mg/kg intravenous injection of an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, may be given as a bolus, and the restof the antibody dose may be administered by intravenous injection. Apredetermined dose of an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, may be administered, for example, over a period of anhour and a half to two hours to five hours.

With regard to a therapeutic agent, where the agent is, e.g., a smallmolecule, it can be present in a pharmaceutical composition in the formof a physiologically acceptable ester or salt, such as in combinationwith a physiologically acceptable cation or anion, as is well known inthe art.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

In one embodiment the compositions of the disclosure are pyrogen-freeformulations which are substantially free of endotoxins and/or relatedpyrogenic substances. Endotoxins include toxins that are confined insidea microorganism and are released when the microorganisms are broken downor die. Pyrogenic substances also include fever-inducing, thermostablesubstances (glycoproteins) from the outer membrane of bacteria and othermicroorganisms. Both of these substances can cause fever, hypotensionand shock if administered to humans. Due to the potential harmfuleffects, it is advantageous to remove even low amounts of endotoxinsfrom intravenously administered pharmaceutical drug solutions. The Foodand Drug Administration (“FDA”) has set an upper limit of 5 endotoxinunits (EU) per dose per kilogram body weight in a single one hour periodfor intravenous drug applications (The United States PharmacopeialConvention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeuticproteins are administered in amounts of several hundred or thousandmilligrams per kilogram body weight it is advantageous to remove eventrace amounts of endotoxin. In one embodiment, endotoxin and pyrogenlevels in the composition are less than 10 EU/mg, or less than 5 EU/mg,or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg,or less than 0.001 EU/mg. In another embodiment, endotoxin and pyrogenlevels in the composition are less than about 10 EU/mg, or less thanabout 5 EU/mg, or less than about 1 EU/mg, or less than about 0.1 EU/mg,or less than about 0.01 EU/mg, or less than about 0.001 EU/mg.

In one embodiment, the disclosure comprises administering a compositionwherein said administration is oral, parenteral, intramuscular,intranasal, vaginal, rectal, lingual, sublingual, buccal, intrabuccal,intravenous, cutaneous, subcutaneous or transdermal.

In another embodiment the disclosure further comprises administering acomposition in combination with other therapies, such as surgery,chemotherapy, hormonal therapy, biological therapy, immunotherapy orradiation therapy.

VI. DOSING/ADMINISTRATION

To prepare pharmaceutical or sterile compositions including an anti-αvβ8integrin antibody, or antigen-binding fragment thereof of thedisclosure, the antibody is mixed with a pharmaceutically acceptablecarrier or excipient. Formulations of therapeutic and diagnostic agentscan be prepared by mixing with physiologically acceptable carriers,excipients, or stabilizers in the form of, e.g., lyophilized powders,slurries, aqueous solutions, lotions, or suspensions (see, e.g.,Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis ofTherapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: TheScience and Practice of Pharmacy, Lippincott, Williams, and Wilkins, NewYork, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:Parenteral Medications, Marcel Dekker, N.Y.; Lieberman, et al. (eds.)(1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, N.Y.;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: DisperseSystems, Marcel Dekker, N.Y.; Weiner and Kotkoskie (2000) ExcipientToxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. In certainembodiments, an administration regimen maximizes the amount oftherapeutic delivered to the patient consistent with an acceptable levelof side effects. Accordingly, the amount of biologic delivered dependsin part on the particular entity and the severity of the condition beingtreated. Guidance in selecting appropriate doses of antibodies,cytokines, and small molecules are available (see, e.g., Wawrzynczak,1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK;Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis,Marcel Dekker, New York, N.Y.; Bach (ed.), 1993, Monoclonal Antibodiesand Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York,N.Y.; Baert, et al., 2003, New Engl. J. Med. 348:601-608; Milgrom, etal., 1999, New Engl. J. Med. 341:1966-1973; Slamon, et al., 2001, NewEngl. J. Med. 344:783-792; Beniaminovitz, et al., 2000, New Engl. J.Med. 342:613-619; Ghosh, et al., 2003, New Engl. J. Med. 348:24-32;Lipsky, et al., 2000, New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentdisclosure employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

Compositions comprising anti-αvβ8 integrin antibodies or antigen-bindingfragments thereof, of the disclosure can be provided by continuousinfusion, or by doses at intervals of, e.g., one day, one week, or 1-7times per week. Doses may be provided intravenously, subcutaneously,topically, orally, nasally, rectally, intramuscular, intracerebrally, orby inhalation. A specific dose protocol is one involving the maximaldose or dose frequency that αvoids significant undesirable side effects.A total weekly dose may be at least 0.05 μg/kg body weight, at least 0.2μg/kg, at least 0.5 μg/kg, at least 1 μg/kg, at least 10 μg/kg, at least100 μg/kg, at least 0.2 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg,at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25mg/kg, or at least 50 mg/kg (see, e.g., Yang, et al., 2003, New Engl. J.Med. 349:427-434; Herold, et al., 2002, New Engl. J. Med. 346:1692-1698;Liu, et al., 1999, J. Neurol. Neurosurg. Psych. 67:451-456; Portielji,et al., 2003, Cancer. Immunol. Immunother. 52: 133-144). The dose may beat least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg,at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least80 μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least 100μg. The doses administered to a subject may number at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12, or more.

For anti-αvβ8 integrin antibodies or antigen-binding fragments thereofof the disclosure, the dosage administered to a patient may be 0.0001mg/kg to 100 mg/kg of the patient's body weight. The dosage may bebetween 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kgand 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg,0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight. In someembodiments, a dosage of an anti-αvβ8 integrin antibody orantigen-binding fragment thereof, administered to a patient in needthereof, is about 0.1 mg/kg, about 0.3 mg/kg, about 2 mg/kg or about 3.0mg/kg of the patient's body weight. In some embodiments, a dosage of ananti-αvβ8 integrin antibody or antigen-binding fragment thereof,administered to a patient in need thereof, is about 0.4 mg/kg, about 4mg/kg, about 40 mg/kg or about 100 mg/kg of the patient's body weight.

In some embodiments, a dosage of an anti-αvβ8 integrin antibody orantigen-binding fragment thereof, administered to a patient in needthereof every 14 days, is about 1 mg/kg to about 12 mg/kg of thepatient's body weight. In some embodiments, a dosage of an anti-αvβ8integrin antibody or antigen-binding fragment thereof, administered to apatient in need thereof every 14 days, is about 2 mg/kg of the patient'sbody weight. In some embodiments, a dosage of an anti-αvβ8 integrinantibody or antigen-binding fragment thereof, administered to a patientin need thereof every 14 days, is about 7 mg/kg of the patient's bodyweight.

In some embodiments, a dosage of an anti-αvβ8 integrin antibody orantigen-binding fragment thereof, administered to a patient in needthereof every 28 days, is about 1 mg/kg to about 20 mg/kg of thepatient's body weight. In some embodiments, a dosage of an anti-αvβ8integrin antibody or antigen-binding fragment thereof, administered to apatient in need thereof every 28 days, is about 4 mg/kg of the patient'sbody weight. In some embodiments, a dosage of an anti-αvβ8 integrinantibody or antigen-binding fragment thereof, administered to a patientin need thereof every 28 days, is about 12 mg/kg of the patient's bodyweight.

The dosage of an anti-αvβ8 integrin antibodies antibody orantigen-binding fragment thereof may be calculated using the patient'sweight in kilograms (kg) multiplied by the dose to be administered inmg/kg. The dosage of the antibodies of the disclosure may be 150 μg/kgor less, 125 μg/kg or less, 100 μg/kg or less, 95 μg/kg or less, 90μg/kg or less, 85 μg/kg or less, 80 μg/kg or less, 75 μg/kg or less, 70μg/kg or less, 65 μg/kg or less, 60 μg/kg or less, 55 μg/kg or less, 50μg/kg or less, 45 μg/kg or less, 40 μg/kg or less, 35 μg/kg or less, 30μg/kg or less, 25 μg/kg or less, 20 μg/kg or less, 15 μg/kg or less, 10μg/kg or less, 5 μg/kg or less, 2.5 μg/kg or less, 2 μg/kg or less, 1.5μg/kg or less, 1 μg/kg or less, 0.5 μg/kg or less, or 0.1 μg/kg or lessof a patient's body weight.

Unit dose of an anti-αvβ8 integrin antibodies or antigen-bindingfragments thereof of the disclosure may be 0.1 mg to 200 mg, 0.1 mg to175 mg, 0.1 mg to 150 mg, 0.1 mg to 125 mg, 0.1 mg to 100 mg, 0.1 mg to75 mg, 0.1 mg to 50 mg, 0.1 mg to 30 mg, 0.1 mg to 20 mg, 0.1 mg to 15mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg,0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg,0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg. Inone embodiment, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof of the disclosure is administered at a unit dose ofabout 100 mg, about 300 mg, about 500 mg, about 600 mg, about 800 mg,about 1200 mg, about 1400 mg or about 1600 mg.

The dosage of an anti-αvβ8 integrin antibodies or antigen-bindingfragments thereof of the disclosure may achieve a serum titer of atleast 0.1 μg/mL, at least 0.5 μg/mL, at least 1 μg/mL, at least 2 μg/mL,at least 5 μg/mL, at least 6 μg/mL, at least 10 μg/mL, at least 15μg/mL, at least 20 μg/mL, at least 25 μg/mL, at least 50 μg/mL, at least100 μg/mL, at least 125 μg/mL, at least 150 μg/mL, at least 175 μg/mL,at least 200 μg/mL, at least 225 μg/mL, at least 250 μg/mL, at least 275μg/mL, at least 300 μg/mL, at least 325 μg/mL, at least 350 μg/mL, atleast 375 μg/mL, or at least 400 μg/mL in a subject. Alternatively, thedosage of the antibodies of the disclosure may achieve a serum titer ofat least 0.1 μg/mL, at least 0.5 μg/mL, at least 1 μg/mL, at least, 2μg/mL, at least 5 μg/mL, at least 6 μg/mL, at least 10 μg/mL, at least15 μg/mL, at least 20 μg/mL, at least 25 μg/mL, at least 50 μg/mL, atleast 100 μg/mL, at least 125 μg/mL, at least 150 μg/mL, at least 175μg/mL, at least 200 μg/mL, at least 225 μg/mL, at least 250 μg/mL, atleast 275 μg/mL, at least 300 μg/mL, at least 325 μg/mL, at least 350μg/mL, at least 375 μg/mL, or at least 400 μg/mL in the subject.

Doses of anti-αvβ8 integrin antibodies, or antigen-binding fragmentsthereof of the disclosure may be repeated and the administrations may beseparated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days,30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method route and dose of administration and the severity ofside effects (see, e.g., Maynard, et al., 1996, A Handbook of SOPs forGood Clinical Practice, Interpharm Press, Boca Raton, FIa.; Dent, 2001,Good Laboratory and Good Clinical Practice, Urch Publ, London, UK).

The route of administration may be by, e.g., topical or cutaneousapplication, injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial,intracerebrospinal, intralesional, or by sustained release systems or animplant (see, e.g., Sidman et al., 1983, Biopolymers 22:547-556; Langer,et al., 1981, J. Biomed. Mater. Res. 15: 167-277; Langer, 1982, Chem.Tech. 12:98-105; Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA82:3688-3692; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA77:4030-4034; U.S. Pat. Nos. 6,350,466 and 6,316,024). In oneembodiment, an anti-αvβ8 integrin antibody, or antigen-binding fragmentthereof of the disclosure is administered intravenously. In oneembodiment, an anti-αvβ8 integrin antibody, or antigen-binding fragmentthereof of the disclosure is administered subcutaneously.

Where necessary, the composition may also include a solubilizing agentand a local anesthetic such as lidocaine to ease pain at the site of theinjection. In addition, pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903, each of which is incorporated herein byreference their entirety. In one embodiment, an anti-αvβ8 antibody, orantigen-binding fragment thereof, or a composition of the disclosure isadministered using Alkermes AIR™ pulmonary drug delivery technology(Alkermes, Inc., Cambridge, Mass.).

A composition of the present disclosure may also be administered via oneor more routes of administration using one or more of a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. Selected routes of administration for antibodies of thedisclosure include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. Parenteraladministration may represent modes of administration other than enteraland topical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion. Alternatively, a composition of the disclosure can beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

If an anti-αvβ8 integrin antibodies, or antigen-binding fragmentsthereof, of the disclosure are administered in a controlled release orsustained release system, a pump may be used to achieve controlled orsustained release (see, Langer, supra; Sefton, 1987, CRC Crit. Ref.Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:501; Saudek etal., 1989, N. Engl. J. Med. 321:514).

Polymeric materials can be used to achieve controlled or sustainedrelease of the therapies of the disclosure (see e.g., MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, 1983, J., Macromol. ScL Rev. Macromol. Chem. 23:61;see also Levy et al., 1985, Science 11 225:190; During et al., 19Z9,Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71: 105); U.S.Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCTPublication No. WO 99/15154; and PCT Publication No. WO 99/20253.Examples of polymers used in sustained release formulations include, butare not limited to, poly(2-hydroxy ethyl methacrylate), poly(methylmethacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), polyvinyl alcohol), polyacrylamide,polyethylene glycol), polylactides (PLA), polyoeactide-co-glycolides)(PLGA), and polyorthoesters. In one embodiment, the polymer used in asustained release formulation is inert, free of leachable impurities,stable on storage, sterile, and biodegradable. A controlled or sustainedrelease system can be placed in proximity of the prophylactic ortherapeutic target, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer, 1990,Science 249:1527-1533. Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore antibodies of the disclosure or conjugates thereof. See, e.g., U.S.Pat. No. 4,526,938, International Patent Publication Nos. WO 91/05548,WO 96/20698, Ning et al., 1996, “Intratumoral Radioimmunotheraphy of aHuman Colon Cancer Xenograft Using a Sustained-Release Gel,”Radiotherapy and Oncology 59:179-189, Song et al., 1995, “AntibodyMediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal ofPharmaceutical Science and Technology 50:372-397, Cleek et al., 1997,“Biodegradable Polymeric Carriers for a bFGF Antibody for CardiovascularApplication,” Pro. Ml. Symp. Control. Rel. Bioact. Mater. 24:853-854,and Lam et al., 1997, “Microencapsulation of Recombinant HumanizedMonoclonal Antibody for Local Delivery,” Proc. Ml. Symp. Control Rel.Bioact. Mater. 24:759-160, each of which is incorporated herein byreference in their entirety.

If an anti-αvβ8 integrin antibody, or antigen-binding fragment thereof,of the disclosure is administered topically, it can be formulated in theform of an ointment, cream, transdermal patch, lotion, gel, shampoo,spray, aerosol, solution, emulsion, or other form well-known to one ofskill in the art. See, e.g., Remington's Pharmaceutical Sciences andIntroduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co.,Easton, Pa. (1995). For non-sprayable topical dosage forms, viscous tosemi-solid or solid forms comprising a carrier or one or more excipientscompatible with topical application and having a dynamic viscosity, insome instances, greater than water are typically employed. Suitableformulations include, without limitation, solutions, suspensions,emulsions, creams, ointments, powders, liniments, salves, and the like,which are, if desired, sterilized or mixed with auxiliary agents (e.g.,preservatives, stabilizers, wetting agents, buffers, or salts) forinfluencing various properties, such as, for example, osmotic pressure.Other suitable topical dosage forms include sprayable aerosolpreparations wherein the active ingredient, in some instances, incombination with a solid or liquid inert carrier, is packaged in amixture with a pressurized volatile (e.g., a gaseous propellant, such asfreon) or in a squeeze bottle. Moisturizers or humectants can also beadded to pharmaceutical compositions and dosage forms if desired.Examples of such additional ingredients are well-known in the art.

If the compositions comprising anti-αvβ8 integrin antibodies, orantigen-binding fragments thereof, are administered intranasally, it canbe formulated in an aerosol form, spray, mist or in the form of drops.In particular, prophylactic or therapeutic agents for use according tothe present disclosure can be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant (e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas). In the case of a pressurized aerosol the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges (composed of, e.g., gelatin) for use in aninhaler or insufflator may be formulated containing a powder mix of thecompound and a suitable powder base such as lactose or starch.

Methods for co-administration or treatment with an additionaltherapeutic agent, e.g., an immune checkpoint molecule, a cytokine, asteroid, a chemotherapeutic agent, an antibiotic, or a radiationtherapy, are well known in the art (see, e.g., Hardman, et al. (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.) (2001)Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams and Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams andWilkins, Phila., Pa.).

An effective amount of therapeutic may decrease the symptoms by at least10 percent; by at least 20 percent; at least about 30 percent; at least40 percent, or at least 50 percent.

Additional therapies (e.g., prophylactic or therapeutic agents), whichcan be administered in combination with an anti-αvβ8 integrinantibodies, or antigen-binding fragments of the disclosure, may beadministered less than 5 minutes apart, less than 30 minutes apart, 1hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, atabout 2 hours to about 3 hours apart, at about 3 hours to about 4 hoursapart, at about 4 hours to about 5 hours apart, at about 5 hours toabout 6 hours apart, at about 6 hours to about 7 hours apart, at about 7hours to about 8 hours apart, at about 8 hours to about 9 hours apart,at about 9 hours to about 10 hours apart, at about 10 hours to about 11hours apart, at about 11 hours to about 12 hours apart, at about 12hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apartfrom the antibodies of the disclosure. The two or more therapies may beadministered within one same patient visit.

Methods of administering the antibody molecules are known in the art andare described below. Suitable dosages of the molecules used will dependon the age and weight of the subject and the particular drug used.Dosages and therapeutic regimens of an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, antibody molecule can be determined bya skilled artisan. In certain embodiments, an anti-αvβ8 integrinantibody, or antigen-binding fragment thereof, is administered byinjection (e.g., subcutaneously or intravenously) at a dose of about 1to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 10to about 14 mg/k, about 5 to 9 mg/kg, about 7 mg/kg, or about 12 mg/kg.In some embodiments, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, is administered at a dose of about 1 mg/kg, about 2mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about7 mg/kg, about 8 mg/kg, about 9 mg/kg or 10 mg/kg, about 11 mg/kg, about12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg,about 30 mg/kg, or about 40 mg/kg. In some embodiments, an anti-αvβ8integrin antibody, or antigen-binding fragment thereof, is administeredat a dose of about 1-5 mg/kg, about 5-10 mg/kg, or about 10-15 mg/kg. Insome embodiments, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, is administered at a dose of about 0.5-2, 2-4, 2-5,2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 5-15, or 5-20 mg/kg.

The dosing schedule can vary from e.g., once a week to once every 2, 3,4, 5, or 6 weeks. In one embodiment, an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, is administered at a dose from about10 to 20 mg/kg (e.g., about 7 mg/kg or about 12 mg/kg) every other week(e.g., every two weeks or biweekly). In one embodiment, an anti-αvβ8integrin antibody, or antigen-binding fragment thereof, is administeredat a dose from about 10 to 20 mg/kg (e.g., about 7 mg/kg or about 12mg/kg) once per month (e.g., every four weeks). In some embodiments, ananti-αvβ8 integrin antibody, or antigen-binding fragment thereof, or apharmaceutical composition comprising the same, is administeredintravenously. In some embodiments, an anti-αvβ8 integrin antibody, orantigen-binding fragment thereof, or a pharmaceutical compositioncomprising the same, is administered subcutaneously.

In some embodiments, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof of the disclosure is administered intravenously orsubcutaneously on a biweekly basis. In one embodiment, an anti-αvβ8integrin antibody, or antigen-binding fragment thereof of the disclosureis administered at a unit dose of about 100 mg, about 300 mg, about 800mg, about 1200 mg or about 1600 mg intravenously or subcutaneously on abiweekly basis. In some embodiments, a subject is administered ananti-αvβ8 integrin antibody, or antigen-binding fragments thereof of thedisclosure at a unit dose of about 100 mg, about 300 mg, about 800 mg,about 1200 mg or about 1600 mg intravenously on a biweekly basis andadministered an anti-PD1 inhibitor every four weeks. In someembodiments, an anti-PD1 inhibitor is an antibody administeredsubcutaneously at a unit dose of 300 mg. In some embodiments, ananti-PD1 inhibitor is an anti-PD1 antibody described in PCT PublicationNo. WO2016/092419 (e.g., mAb7, also referred to as RN888, PF-06801591,or sasanlimab).

In some embodiments, an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, or a pharmaceutical composition comprising the same,is administered about twice a week, once a week, once every two weeks,once every three weeks, once every four weeks, once every five weeks,once every six weeks, once every seven weeks, once every eight weeks,once every nine weeks, once every ten weeks, twice a month, once amonth, once every two months, once every three months, once every fourmonths, once every five months, once every six months, once every sevenmonths, once every eight months, once every nine months, once every tenmonths, once every eleven months or once every twelve months. In someembodiments, an anti-αvβ8 integrin antibody, or antigen-binding fragmentthereof, or a pharmaceutical composition comprising the same, isadministered is administered every two weeks, e.g., up to 12 times(e.g., up to 10, 8, 6, 5, 4, or 3 times). In some embodiments, ananti-αvβ8 integrin antibody, or antigen-binding fragment thereof, or apharmaceutical composition comprising the same, is administered isadministered every four weeks, e.g., up to 12 times (e.g., up to 10, 8,6, 5, 4, or 3 times).

In some embodiments, each administration of an anti-αvβ8 integrinantibody, or antigen-binding fragment thereof, comprises 5-10 mg/kg(e.g., 5, 6, 7, 8, 9, or 10 mg/kg) of the antibody, or theantigen-binding fragments thereof, e.g., each administration comprisesabout 7 mg/kg.

In some embodiments, the antibody, or an antigen-binding fragmentsthereof, is administered every four weeks, e.g., up to 6 times (e.g., upto 6, 5, 4, 3, 2, or 1 time).

In other embodiments, each administration of an anti-αvβ8 integrinantibody, or antigen-binding fragment thereof, comprises 10-15 mg/kg(e.g., 10, 11, 12, 13, 14, or 15 mg/kg) of the antibody, or theantigen-binding fragments thereof, e.g., each administration comprisesabout 12 mg/kg.

Anti-αvβ8 antibodies, or antigen-binding fragments thereof, of thedisclosure and other therapies may be cyclically administered. Cyclingtherapy involves the administration of a first therapy (e.g., a firstprophylactic or therapeutic agent) for a period of time, followed by theadministration of a second therapy (e.g., a second prophylactic ortherapeutic agent) for a period of time, optionally, followed by theadministration of a third therapy (e.g., prophylactic or therapeuticagent) for a period of time and so forth, and repeating this sequentialadministration, i.e., the cycle in order to reduce the development ofresistance to one of the therapies, to αvoid or reduce the side effectsof one of the therapies, and/or to improve the efficacy of thetherapies.

In certain embodiments, anti-αvβ8 antibodies, or antigen-bindingfragments thereof of the disclosure can be formulated to ensure properdistribution in vivo. For example, the blood-brain barrier (BBB)excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the disclosure cross the BBB (if desired), theycan be formulated, for example, in liposomes. For methods ofmanufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;and 5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., V. V. Ranade, 1989, J. Clin.Pharmacol. 29:685). Exemplary targeting moieties include folate orbiotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al.,Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman etal., 1995, FEBS Lett. 357: 140; M. Owais et al., 1995, Antimicrob.Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe etal. (1995) Am. J. Physiol. 1233: 134); p120 (Schreier et al. (1994) J.Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen, 1994, FEBSLett. 346:123; Killion; Fidler, 1994; Immunomethods 4:273.

The disclosure provides protocols for the administration ofpharmaceutical composition comprising anti-αvβ8 antibodies, orantigen-binding fragments thereof, of the disclosure alone or incombination with other therapies to a subject in need thereof. Thetherapies (e.g., prophylactic or therapeutic agents) of the combinationtherapies of the present disclosure can be administered concomitantly orsequentially to a subject. The therapy (e.g., prophylactic ortherapeutic agents) of the combination therapies of the presentdisclosure can also be cyclically administered.

The therapies (e.g., prophylactic or therapeutic agents) of thecombination therapies of the disclosure can be administered to a subjectconcurrently. The term “concurrently” is not limited to theadministration of therapies (e.g., prophylactic or therapeutic agents)at exactly the same time, but rather it is meant that a pharmaceuticalcomposition comprising anti-αvβ8 integrin antibodies, or antigen-bindingfragments thereof, of the disclosure are administered to a subject in asequence and within a time interval such that the antibodies of thedisclosure or conjugates thereof can act together with the othertherapy(ies) to provide an increased benefit than if they wereadministered otherwise. For example, each therapy may be administered toa subject at the same time or sequentially in any order at differentpoints in time; however, if not administered at the same time, theyshould be administered sufficiently close in time so as to provide thedesired therapeutic or prophylactic effect. Each therapy can beadministered to a subject separately, in any appropriate form and by anysuitable route. In various embodiments, the therapies (e.g.,prophylactic or therapeutic agents) are administered to a subject lessthan 15 minutes, less than 30 minutes, less than 1 hour apart, at about1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hoursto about 3 hours apart, at about 3 hours to about 4 hours apart, atabout 4 hours to about 5 hours apart, at about 5 hours to about 6 hoursapart, at about 6 hours to about 7 hours apart, at about 7 hours toabout 8 hours apart, at about 8 hours to about 9 hours apart, at about 9hours to about 10 hours apart, at about 10 hours to about 11 hoursapart, at about 11 hours to about 12 hours apart, 24 hours apart, 48hours apart, 72 hours apart, or 1 week apart. In other embodiments, twoor more therapies (e.g., prophylactic or therapeutic agents) areadministered to a within the same patient visit.

The prophylactic or therapeutic agents of the combination therapies canbe administered to a subject in the same pharmaceutical composition.Alternatively, the prophylactic or therapeutic agents of the combinationtherapies can be administered concurrently to a subject in separatepharmaceutical compositions. The prophylactic or therapeutic agents maybe administered to a subject by the same or different routes ofadministration.

VII. KITS

The disclosure also provides kits comprising any or all of theantibodies described herein. Kits of the disclosure include one or morecontainers comprising an anti-αvβ8 integrin antibody, or antigen-bindingfragment thereof, described herein and instructions for use inaccordance with any of the methods of the disclosure described herein.Generally, these instructions comprise a description of administrationof an antibody for the above described therapeutic treatments. In someembodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing anapplicator, e.g., single and multi-chambered pre-filled syringes (e.g.,liquid syringes and lyosyringes), are included.

The instructions relating to the use of an anti-αvβ8 integrin antibody,or antigen-binding fragment thereof, generally include information as todosage, dosing schedule, and route of administration for the intendedtreatment. The containers may be unit doses, bulk packages (e.g.,multi-dose packages) or sub-unit doses. Instructions supplied in thekits of the disclosure are typically written instructions on a label orpackage insert (e.g., a paper sheet included in the kit), butmachine-readable instructions (e.g., instructions carried on a magneticor optical storage disk) are also acceptable.

The kits of this disclosure are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Also contemplated are packages for use in combination with a specificdevice, such as an inhaler, nasal administration device (e.g., anatomizer) or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an anti-αvβ8 integrin antibody ofthe disclosure. The container may further comprise an additionaltherapeutic agent as described herein.

The kit may further comprise at least one anti-PD1 antibody, such as,but not limited to, nivolumab, pembrolizumab, spartlizumab, pidilizumab,tislelizumab, cemiplimab, sasanlimab (mAb7, RN888, PD-06801591),AMP-224, an AMP-514.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

The disclosure also provides diagnostic kits comprising any or all ofthe antibodies, or antigen-binding fragments thereof, described herein.The diagnostic kits are useful for, for example, detecting the presenceof αvβ8 integrin in a sample. In some embodiments, a diagnostic kit canbe used to identify an individual with a latent disease, disorder orcondition that may put them at risk of developing αvβ8 integrin-mediateddisease, disorder or condition or a αvβ8 integrin deficiency disease,disorder or condition. In some embodiments, a diagnostic kit can be usedto detect the presence and/or level of αvβ8 integrin in an individualsuspected of having a αvβ8 integrin mediated disease or a αvβ8 integrindeficiency disease, disorder or condition.

Diagnostic kits of the disclosure include one or more containerscomprising an anti-αvβ8 integrin antibody, or antigen-binding fragmentthereof, described herein and instructions for use in accordance withany of the methods of the disclosure described herein. Generally, theseinstructions comprise a description of use of an anti-αvβ8 integrinantibody, or antigen-binding fragment thereof, to detect the presence ofαvβ8 integrin in individuals at risk for, or suspected of having, a αvβ8integrin mediated disease or a αvβ8 integrin deficiency disease,disorder or condition. In some embodiments, an exemplary diagnostic kitcan be configured to contain reagents such as, for example, an anti-αvβ8integrin antibody, or antigen-binding fragment thereof, a negativecontrol sample, a positive control sample, and directions for using thekit.

VIII. EQUIVALENTS

The foregoing description and following Examples detail certain specificembodiments of the disclosure and describes the best mode contemplatedby the inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the disclosure may bepracticed in many ways and the disclosure should be construed inaccordance with the appended claims and any equivalents thereof.

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications can be made withoutdeparting from the teachings herein and the claimed disclosure below.The following examples are provided to better illustrate the disclosedteachings and are not intended to limit the scope of the teachingspresented herein. While the present teachings have been described interms of these exemplary embodiments, the skilled artisan will readilyunderstand that numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

IX. GENERAL TECHNIQUES

It is to be understood that this invention is not limited to specificsynthetic methods of making that may of course vary. Unless otherwisedefined herein, scientific and technical terms used in connection withthe present invention shall have the meanings that are commonlyunderstood by those of ordinary skill in the art. Further, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. Generally, nomenclaturesused in connection with, and techniques of, cell and tissue culture,molecular biology, immunology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Sambrook and Russell, MolecularCloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001); Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, N Y (2002); Harlowand Lane Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1998); Coligan et al., ShortProtocols in Protein Science, John Wiley & Sons, N Y (2003); ShortProtocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean,eds., Oxford University Press, 2000); Using antibodies: a laboratorymanual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press,1999); The Antibodies (M. Zanetti and J. D. Capra, eds., HarwoodAcademic Publishers, 1995).

Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry,biochemistry, immunology, molecular biology, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well-known and commonly used in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

X. BIOLOGICAL DEPOSIT

Representative materials of the present invention were deposited in theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209, USA, on Feb. 13, 2018. Vector ADWA11 VH05-02-VH, havingATCC Accession No. PTA-124917, comprises a DNA insert encoding the heavychain variable region of antibody ADWA11 2.4, also known as VH05-2VK01(2.4) and ADWA11 5-2 2.4. Vector ADWA11 VK2.4-VL, having ATCCAccession No. PTA-124918, comprises a DNA insert encoding the lightchain variable region of antibody ADWA11 2.4, also known as VH05-2VK01(2.4) and ADWA11 5-2 2.4.

The deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Pfizer Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the culture of the deposit to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. Section 122 and the Commissioner's rules pursuant thereto(including 37 C.F.R. Section 1.14 with particular reference to 886 OG638).

The owner of the present application has agreed that if a culture of thematerials on deposit should die or be lost or destroyed when cultivatedunder suitable conditions, the materials will be promptly replaced onnotification with another of the same. Availability of the depositedmaterial is not to be construed as a license to practice the inventionin contravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

EXAMPLES

The disclosure is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the disclosure should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1: Generation of Anti-αvβ8 Integrin Mouse Hybridoma Antibodies

Mouse hybridoma antibodies against human αvβ8 integrin were generatedaccording to the methods as generally described in U.S. Pat. No.9,969,804, which is herein incorporated by reference in its entirety.

Briefly, integrin (38 knockout mice, which were crossed into the outbredCD1 background to permit post-natal survival, were immunized withrecombinant human αvβ8 integrin (R&D Systems, 4135-AV-050) at a dosageof 50 μg per mouse every two weeks until acceptable titers of anti-αvβ8antibodies were generated. The sera from immunized mice were thenscreened by solid phase immunoassay to identify mice for hybridomageneration.

Antibodies from generated hybridomas were further characterized by flowcytometry using SW480 cells transfected to express integrin αvβ8 or αvβ3or αvβ6 as negative controls. Sw480 cells normally do not express any αvintegrins except for αvβ5. The mouse hybridoma antibody ADWA-11 (alsoreferred to as ADWA11) was identified, and to confirm the specificity ofthis antibody flow cytometry was performed on each cell line usinglabelled ADWA-11 or antibodies to αvβ5 (Alula) or αvβ3 (Axum-2) or αvβ6(10D5) (Su et al., Am. J. Respir. Cell Mol. Biol. 36:377-386, 2007; Suet al., Am. J. Respir. Cell Mol. Biol. 185: 58-66, 2012; Huang et al.,J. Cell Sci. 111 (Pt 15): 2189-2195).

Cell adhesion assays were also performed with U251 cells that expressintegrin αvβ8 on dishes coated with TGFβ1 latency associated peptide 1μg/ml (Kueng et al., Anal. Biochem. 182: 16-19, 1989). Blockade of TGFβactivity was determined by TMLC luciferase assay, which utilizes minklung epithelial cells expressing a TGFβ sensitive portion of PAI-1promoter driving firefly luciferase expression (Abe et al., Anal.Biochem. 216:276-284, 1994). Based on the hybridoma screening performedgenerally as described herein, the mouse hybridoma antibody ADWA-11 wasselected for further evaluation.

Example 2: Humanization of Anti-αvβ8 Integrin Mouse Hybridoma Antibodies

The mouse hybridoma antibody ADWA-11, as disclosed in U.S. Pat. No.9,969,804, and as set forth in, e.g., SEQ ID NO: 20-33 and 71-76 of thepresent description, was humanized by grafting the murine CDR sequencesinto the various human germline frameworks as listed in Table 1, whichincluded the light chain germline frameworks IGKV2-28, IGKV2-30,IGKV4-1, IGKV1-39 (also referred to herein as DPK9), and IGKV3-11, aswell as the heavy chain germline frameworks IGHV3-7 (also referred toherein as DP54), IGHV1-46, IGHV3-23, IGHV3-30, IGHV1-69, and IGHV3-48(see, e.g., IMGT database).

The humanized antibodies referred to herein as “Humanized ADWA-11”included a set of six murine CDR sequences as set forth in SEQ ID NOs:20-33 and 71-76 grafted into a IGKV1-39 (e.g., DPK9) light chaingermline framework and a IGHV3-7 (e.g., DP54) heavy chain germlineframework. Other germlines framework variants were also tried, as shownbelow in Tables 1.1 and 1.2.

TABLE 1.1 Humanized variant SEP ID NO ADWA11 IGHV1- VH: SEQ ID NO: 34;46/IGKV1-39 VL: SEQ ID NO: 65 ADWA11 IGHV1- VH: SEQ ID NO: 34;46/IGKV2-28 VL: SEQ ID NO: 62 ADWA11 IGHV1- VH: SEQ ID NO: 34;46/IGKV3-11 VL: SEQ ID NO: 66 ADWA11 IGHV1 - VH: SEQ ID NO: 34;46/IGKV2-30 VL: SEQ ID NO: 63 ADWA11 IGHV1- VH: SEQ ID NO: 34;46/IGKV4-1 VL: SEQ ID NO: 64 ADWA11 IGHV1- VH: SEQ ID NO: 37;69/IGKV1-39 VL: SEQ ID NO: 65 ADWA11 IGHV1- VH: SEQ ID NO: 37;69/IGKV2-28 VL: SEQ ID NO: 62 ADWA11 IGHV1- VH: SEQ ID NO: 37;69/IGKV3-11 VL: SEQ ID NO: 66 ADWA11 IGHV1- VH: SEQ ID NO: 37;69/IGKV2-30 VL: SEQ ID NO: 63 ADWA11 IGHV1- VH: SEQ ID NO: 37;69/IGKV4-1 VL: SEQ ID NO: 64 ADWA11 IGHV3- VH: SEQ ID NO: 36;30/IGKV1-39 VL: SEQ ID NO: 65 ADWA11 IGHV3- VH: SEQ ID NO: 36;30/IGKV2-28 VL: SEQ ID NO: 62 ADWA11 IGHV3- VH: SEQ ID NO: 36;30/IGKV3-11 VL: SEQ ID NO: 66 ADWA11 IGHV3- VH: SEQ ID NO: 36;30/IGKV2-30 VL: SEQ ID NO: 63 ADWA11 IGHV3- VH: SEQ ID NO: 36;30/IGKV4-1 VL: SEQ ID NO: 64 ADWA11 IGHV3- VH: SEQ ID NO: 35;23/IGKV1-39 VL: SEQ ID NO: 65 ADWA11 IGHV3- VH: SEQ ID NO: 35;23/IGKV2-28 VL: SEQ ID NO: 62 ADWA11 IGHV3- VH: SEQ ID NO: 35;23/IGKV3-11 VL: SEQ ID NO: 66 ADWA11 IGHV3- VH: SEQ ID NO: 35;23/IGKV2-30 VL: SEQ ID NO: 63 ADWA11 IGHV3- VH: SEQ ID NO: 35;23/IGKV4-1 VL: SEQ ID NO: 64 ADWA11 IGHV3- VH: SEQ ID NO: 38;48/IGKV1-39 VL: SEQ ID NO: 65 ADWA11 IGHV3- VH: SEQ ID NO: 38;48/IGKV2-28 VL: SEQ ID NO: 62 ADWA11 IGHV3- VH: SEQ ID NO: 38;48/IGKV3-11 VL: SEQ ID NO: 66 ADWA11 IGHV3- VH: SEQ ID NO: 38;48/IGKV2-30 VL: SEQ ID NO: 63 ADWA11 IGHV3- VH: SEQ ID NO: 38;48/IGKV4-1 VL: SEQ ID NO: 64 ADWA11 chimeric control

TABLE 1.2 Corresponding SEQ ID Name germline NO: Sequence ADWA11IGHV1-46 34 QVQLVQSGAEVKKPGASVKVSCK VH1 ASGFNIKDYYMNWVRQAPGQGLEWIGWIDPDNGNTIYDQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAV YYCARRLLMDYWGQGTLVTVSSADWA11 IGHV3-23 35 EVQLLESGGGLVQPGGSLRLSCAA VH2 SGFNIKDYYMNWVRQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAV YYCARRLLMDYWGQGTLVTVSSADWA11 IGHV3-30 36 QVQLVESGGGVVQPGRSLRLSCA VH3 ASGFNIKDYYMNWVRQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAV YYCARRLLMDYWGQGTLVTVSSADWA11 IGHV1-69 37 QVQLVQSGAEVKKPGSSVKVSCK VH4 ASGFNIKDYYMNWVRQAPGQGLEWIGWIDPDNGNTIYDQKFQGRVTI TADESTSTAYMELSSLRSEDTAVYY CARRLLMDYWGQGTLVTVSSADWA11 IGHV3-48 38 EVQLVESGGGLVQPGGSLRLSCAA VH5 SGFNIKDYYMNWVRQAPGKGLEWIGWIDPDNGNTIYDDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAV YYCARRLLMDYWGQGTLVTVSSADWA11 IGKV2-28 62 DIVMTQSPLSLPVTPGEPASISCRST VK1KSLLHFNGNTYLFWYLQKPGQSP QLLIYYMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSL EYPFTFGQGTKVEIK ADWA11 IGKV2-30 63DVVMTQSPLSLPVTLGQPASISCRS VK2 TKSLLHFNGNTYLFWFQQRPGQSPRRLIYYMSNLASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCMQSL EYPFTFGQGTKVEIK ADWA11IGKV4-1 64 DIVMTQSPDSLAVSLGERATINCRS VK3 TKSLLHFNGNTYLFWYQQKPGQPPKLLIYYMSNLASGVPDRFSGSGS GTDFTLTISSLQAEDVAVYYCMQS LEYPFTFGQGTKVEIKADWA11 IGKV1-39 65 DIQMTQSPSSLSASVGDRVTITCRS VK4 TKSLLHFNGNTYLFWYQQKPGKAPKLLIYYMSNLASGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCMQSL EYPFTFGQGTKVEIKADWA11 IGKV3-11 66 EIVLTQSPATLSLSPGERATLSCRST VK5KSLLHFNGNTYLFWYQQKPGQAP RLLIYYMSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCMQSLEY PFTFGQGTKVEIK

A sequence alignment comparing the heavy chain variable region and thelight chain variable region amino acid sequences of the mouse hybridomaantibody ADWA-11 (referred to as “Hybridoma mADWA-11” and “mADWA11”),the Humanized ADWA-11 antibody (“huADWA11-2.4”), and the IGHV3-07 andIGKV1-39 germline sequences is shown in FIGS. 1A and 1B. The underlinedamino acid residues are the CDR sequences according to Kabat and thebolded amino acid residues are the CDR sequences according to Chothia.

Example 3: Introduction of Framework Mutations into Humanized Anti-αv138Integrin Antibodies

In order to improve the binding affinity of the humanized ADWA-11antibody for αvβ8 integrin as compared to the binding affinity of themouse hybridoma antibody ADWA11, several variants were generated havingframework substitutions in the heavy chain variable region (VH) and thelight chain variable region (VL), as indicated in Table 2. The heavychain residues described in this Example are numbered according to SEQID NO: 127. The light chain residues described in this Example arenumbered according to SEQ ID NO: 128.

TABLE 2 Framework substitutions introduced into the humanized ADWA11antibody Amino acid substitution(s) relative to SEQ ID NO: 127 (forheavy chain Variant ADWA11 Alternative residues) and SEQ ID NO: VH andVL regions Name(s) 128 (for light chain residues) adwaVHl. 1 ADWA11 VH01T28N, F29I (SEQ ID NO: 88) adwa_VH_1.2 ADWA11 VH02 T28N, F29I, R72A (SEQID NO: 89) adwa_VH_1.3 ADWA11 VH03 T28N, F29I, R72A, (SEQ ID NO: 90)A49G, L79A adwa_VH_1.4 ADWA11 VH04 T28N, F29I, R72A, (SEQ ID NO: 91)N74T, A75S adwa_VH_1.5 ADWA11 VH05 T28N, F29I, R72A, A49G, (SEQ ID NO:39) ADWA11 VH05 VK1 L79A,N74T, A75S adwaVLl. 1 ADWA11 VK01 L46R (SEQ IDNO: 47) ADWA11_VK01 (1) adwa_VL_l .2 ADWA11 VK02 L46R, Y36F (SEQ ID NO:92)

Humanized ADWA11 antibody variants were generated by combining thevarious sets of framework substitutions listed in Table 2. Suchcombinations resulted in antibodies having the VH of ADWA11 VH01, ADWA11VH02, ADWA11 VH03, ADWA11 VH04, or ADWA11 VH05, and the VL of eitherADWA11 VK01 or ADWA11 VK02.

The humanized ADWA11 antibody variants ADWA11 VH01/VK01, ADWA11VH02/VK01, ADWA11 VH03/VK01, ADWA11 VH03/VK02, and ADWA11 VH05/VK01 weregenerated and evaluated as generally described herein.

The humanized ADWA11 antibody variant designated ADWA11 VH01/VK01included T28N and F29I substitutions in the VH (SEQ ID NO:88), and anL46R substitution in the VL (SEQ ID NO:47).

The humanized ADWA11 antibody variant ADWA11 VH02/VK01 included T28N,F29I, and R72A substitutions in the VH (SEQ ID NO:89), and an L46Rsubstitution in the VL (SEQ ID NO:47).

The humanized ADWA11 antibody variant ADWA11 VH03/VK01 included T28N,F29I, R72A, A49G, and L79A substitutions in the VH (SEQ ID NO:90), andan L46R substitution in the VL (SEQ ID NO:47).

The humanized ADWA11 antibody variant ADWA11 VH03/VK02 included T28N,F29I, R72A, A49G, and L79A substitutions in the VH (SEQ ID NO:90), andL46R and Y36F substitutions in the VL (SEQ ID NO:92).

The humanized ADWA11 antibody variant ADWA11 VH05/VK01 included T28N,F29I, R72A, A49G, L79A, N74T, and A75S substitutions in the VH (SEQ IDNO: 39), and an L46R substitution in the VL (SEQ ID NO: 47).

The Fab binding affinities of ADWA11 VH01/VK01, ADWA11 VH02/VK01, ADWA11VH03/VK01, ADWA11 VH03/VK02, and ADWA11 VH05/VK01 for αvβ8 integrin weredetermined and are listed in Table 3. Additionally, the IC50 values forinhibiting TGF-β activation were also determined for each antibody andare listed in Table 3.

TABLE 3 Characterization of humanized ADWA11 antibodies variants IgGPotency TGFB Fab Affinity Assay Antibody Name(s) ka kd KD (M) KD (PM)IC50 (pM) Mouse hybridoma antibody 9.22E+04 4.86E−05 5.36E−10  536  183ADWA-11 (n = 3) VH: SEQ ID NO: 20 VL: SEQ ID NO: 21 Also referred to as:Hybridoma mADWA-11 Humanized ADWA11 VH01/VK01 4.13E+04 2.51E−04 6.86E−096860 8473 Variants VH: SEQ ID NO: 88 (n = 2) (DP54/DPK9) VL: SEQ ID NO:47 Also referred to as: VH01/VK01 Fab ADWA11 VH02/VK01 1.30E+05 2.44E−041.89E−09 1890 1756 VH: SEQ ID NO: 89 (n = 2) VL: SEQ ID NO: 47 Alsoreferred to as: VH02/VK01 Fab ADWA11 VH03/VK01 1.36E+05   <1E−057.35E−11 <100  138 VH: SEQ ID NO: 90 VL: SEQ ID NO: 47 Also referred toas: VH03/VK01 Fab ADWA11 VH03/VK02 1.40E+05 5.23E−05 3.73E−10  370  223VH: SEQ ID NO: 90 VL: SEQ ID NO: 92 Also referred to as: VH03/VK02 FabADWA11 VH05/VK01 1.87E+05   <1E−05 5.00E−11 <100  148 VH: SEQ ID NO: 39(n = 3) VL: SEQ ID NO: 47 Also referred to as: VH05-2_VK01; VH05-2_VK01parental; and VH05/VK01 Fab

As shown in Table 3, ADWA11 VH03/VK01, ADWA11 VH03/VK02, and ADWA11VH05/VK01 each demonstrated an improved binding affinity for αvβ8integrin as compared to the mouse hybridoma antibody ADWA-11. ADWA11VH03/VK01 (GBT) and ADWA11 VH05/VK01 each bound to αvβ8 integrin with aKD of less than 100 pM, which represented at least a 5-fold lower KDvalue than the KD of 536 pM determined for the mouse hybridoma antibodyADWA-11. Further, ADWA11 VH03/VK01 and ADWA11 VH05/VK01 eachdemonstrated a lower IC50 value for inhibiting TGF-β transactivation, ascompared to the mouse hybridoma antibody ADWA-11.

It was further determined, as generally described herein, that ADWA11VH05/VK01 also retained the activity, specificity, and species crossreactivity of the mouse hybridoma antibody ADWA-11. Thus, this exampledemonstrates improved binding affinity of humanized ADWA-11 antibodiesfor αvβ8 integrin as compared to the binding affinity of the mousehybridoma antibody ADWA11.

Example 4: Optimization of the Humanized ADWA11 Antibody Variants

Single amino acid substitutions, as listed in Table 4, were evaluatedfor their ability to improve the stability and/or to reduce theimmunogenicity of the humanized antibody ADWA11 VH05/VK01. The heavychain residues described in this Example are numbered according toADWA11 VH05 (SEQ ID NO: 39). The light chain residues described in thisExample are numbered according to ADWA11 VK01 (SEQ ID NO: 47).

TABLE 4 Single Amino Acid Substitutions Light Chain Substitutionsrelative to Ileavy Chain Substitutions relative ADWA11 VK01 (SEQ ID NO:47) to ADWA11 VH05 (SEQ ID NO: 39) L30S K30A Y55A N55Q M56A N57Q N58SD61E A60Q P62A M94Q K63A L97Y F64V F101L F101W Q105G

As shown further here, single substitutions that included K30A, N55Q,N57Q, D61E, or P62A in the variable region of the heavy chain, and L30S,M56A, N58S, M94Q, L97Y, or Q105G in the variable region of the lightchain were found to retain the activity of the parental molecule (FIGS.4A-4B). Additionally, combinations of different single amino acidsubstitutions as listed in Table 5 were also evaluated, and heavy chainsequences containing a double mutant including N55Q and D61E, or atriple mutant including N55Q, D61E, and F64V were found to retain theactivity of the parental molecule, as described further herein (FIG.4C).

More specifically, ADWA11 VH05/VK01 variants including the combinationsof amino acid substitutions listed in Table 5, and referred to as ADWA112.1 (“2.1”), ADWA11 2.2 (“2.2”), ADWA11 2.3 (“2.3”), and ADWA11 2.4(“2.4”) were generated. The ADWA11 VH05-2/VK01 variants ADWA11 2.1 andADWA11 2.4 were shown, as described further herein, to have morefavorable expression and activity properties. This example demonstratesthat single amino acid substitutions improved the stability and/orreduced the immunogenicity of the humanized antibody ADWA11 VH05/VK01.

TABLE 5 Combinations of Amino Acid Substitutions Light Ileavy ChainChain Substitutions Substitutions relative to relative to ADWA11 ADWA11VH05 Alternative VK01 (SEQ ID NO: (SEQ ID NO: Clone Name Name(s) 47) 39)2.1 ADWA11 2.1 L30S, M94Q, Q105G N55Q, D61E VH: SEQ ID NO: 6VH05-2_VK01(2.1) VL: SEQ ID NO: 67 2.2 ADWA11 2.2 L30S, L97Y, Q105GN55Q, D61E VH: SEQ ID NO: 6 VH05-2_VK01(2.2) VL: SEQ ID NO: 68 2.3ADWA11 2.3 L30S, M56S, M94Q, N55Q, D61E VH: SEQ ID NO: 6VH05-2_VK01(2.3) Q105G VL: SEQ ID NO: 69 2.4 ADWA11 2.4 L30S, N58S,M94Q, N55Q, D61E VH: SEQ ID NO: 6 VH05-2_VK01(2.4) Q105G VL: SEQ ID NO:7 ADWA11 5-2 2.4 2.1 (F64V) ADWA11 2.1 (F64V) L30S, M94Q, Q105G N55Q,D61E, VH: SEQ ID NO: 93 VHO5-2 (F64V) F64V VL: SEQ ID NO: 67 VKOl (2.1)2.2 (F64V) ADWA11 2.2 (F64V) L30S, L97Y, Q105G N55Q, D61E, VH: SEQ IDNO: 93 VHO5-2 (F64V) F64V VL: SEQ ID NO: 68 VKO 1 (2.2) 2.3 (F64V)ADWA11 2.3 (F64V) L30S, M56S, M94Q, N55Q, D61E, VH: SEQ ID NO: 93 VHO5-2(F64V) Q105G F64V VL: SEQ ID NO: 69 VKOl (2.3) 2.4 (F64V) ADWA11 2.4(F64V) L30S, N58S, M94Q, N55Q, D61E, VH: SEQ ID NO: 93 VH05-2 (F64V)Q105G F64V VL: SEQ ID NO: 7 VKO 1 (2.4)

Example 5: Generation of Anti-αvβ8 Antibodies without Effector Function

Anti-αvβ8 integrin antibodies having reduced Fc-gamma receptor bindingand reduced effector function (e.g., reduced antibody-dependentcell-mediated cytotoxicity (ADCC) and/or reduced complement dependentcytotoxicity (CDC) functions) were generated by subcloning a light chainvariable region and a heavy chain variable region of the invention, andas listed in Table 1, into an immunoglobulin G (IgG) molecule.

To generate mouse antibodies without effector function, the variableregions from the mouse hybridoma antibody ADWA-11 were subcloned into amouse IgG1 Fc backbone that contained E233P, E318A, K320A, and R322Asingle amino acid substitutions as outlined in U.S. Publication No.US2009/0155256. This antibody is referred to herein as ADWA-11_4mut, andas mIgG_4mut. ADWA-11_4mut binding to mouse αvB8 was assessed using C8-Smouse astrocyte cells (ATCC) and blockade of TGFB activation wasdetermined using C8-S cells in co-culture TMLC luciferase assay.

To generate human antibodies without effector function, the variableregions from the humanized antibody ADWA11 VH05VK01 were subcloned intoa human IgG1 Fc backbone (e.g., as described herein) that containedL234A, L235A, and G237A single amino acid substitutions in the hingeregion, such that the hinge region comprised the amino acid sequenceEPKSCDKTHTCPPCPAPEAAGAP (SEQ ID NO: 126) instead of the wild type hingeregion amino acid sequence EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 125).This human monoclonal antibody without effector function is referred toherein as hIgG_VH05VK01.

Various features of the light chain (SEQ ID NO: 123) and heavy chain(SEQ ID NO: 124) of hIgG_VH05VK01 are identified in the sequences below.

Light Chain of ADWA11 VK01 (SEQ ID NO: 123)DIQMTQSPSSLSASVGDRVTITCRSTKSLLHFNGNTYLFWYQQKPGKAPKRLIYYMSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMQSLEYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy Chain of ADWA11 VH05 (SEQ ID NO: 124)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYMNWVRQAPGKGLEWVGWIDPDNGNTIYDPKFQGRFTISADTSKNSAYLQMNSLRAEDTAVYYCARRLLMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

In the above light chain and heavy chain sequences of humanizedADWA11VH05VK01, the CDR sequences are underlined; the N-linkedglycosylation consensus sequence site is at amino acid residues N295,5296, and T297 of the heavy chain; a potential cleavage site is at aminoacid residues D52 and P53 of the heavy chain; potential deamidationsites are at amino acid residues N33, G34, N35, and T36 of the lightchain, and amino acid residues N57, T58, N77, S78, N84, and S85 of theheavy chain; a potential isomerization site is at amino acid residuesD90 and T91 of the heavy chain; a potential methionine oxidation site isat amino acid residues M4 and M56 of the light chain, and M34, M102, andM426 of the heavy chain; the triple alanine mutant is at amino acidresidues A232 A233, and A235 of the heavy chain; and the non-humanresidues outside the CDRs are amino acid residues R51 of the lightchain, and G49, A72, T74, S75, and A79 of the heavy chain. The residuesin the heavy chain and the light chain described in this Example arenumbered according to SEQ ID NO: 124 and SEQ ID NO: 123, respectively.

Example 6: Evaluation of Anti-αvβ8 Integrin Antibodies by ELISA ELISAMethod

Biotinylated human αvβ8 integrin (50 μl of 0.6 μg/ml) was captured ontoan ELISA plate coated with streptavidin. After blocking and washing,antibodies of interest (e.g., murine, chimera, or humanized variants)were added at various dilutions to different wells and incubated at roomtemperature for 1 hour. After washing, the detection antibody,anti-human IgG-HRP was added and incubated for 1 hour at roomtemperature. After washing, enzyme substrate (TMB) was added to developthe color for 10 minutes. The enzyme reaction was quenched by additionof 0.16 M sulfuric acid and the final signal intensity was measured at450 nm.

Results

Mouse hybridoma antibodies confirmed to bind human αvβ8 (hαvβ8) integrinwere counter screened for binding against the closely related integrinshuman αvβ3 and human αvβ6 to select murine antibodies that bindspecifically to hαvβ8. The mouse hybridoma antibody ADWA11 was specificfor hαvβ8 integrin and did not bind to the closely related integrinshαvβ3 or hαvβ6 (FIG. 2). However, the humanized ADWA11 antibody ADWA11VH05/VK01 showed substantially improved affinity for αvβ8 integrincompared to the mouse hybridoma antibody ADWA11 (FIGS. 3A-3B).

Anti-αvβ8 integrin Fab molecules of ADWA11 VH05/VK01 having single orcombinations of amino acids substitutions, as listed in Tables 4 and 5,were also evaluated by ELISA for binding to hαvβ8 integrin (FIGS.4A-4C). As shown in FIG. 4A, Fabs having a K30A, N55Q, N57Q, D61E, P62A,or K63A single amino acid substitution in the heavy chain variableregion retained binding affinity for hαvβ8. As shown in FIG. 4B, Fabshaving a Y55A, A60Q, F101L, or F101W single amino acid substitution inthe light chain variable region displayed a reduced binding affinity forhαvβ8, as compared to the parental antibody. As shown in FIG. 4C, Fabshaving a combination of amino acid substitutions as listed in Table 5retained binding affinity for hαvβ8.

This example demonstrates that humanized ADWA11 antibody ADWA11VH05/VK01 showed substantially improved affinity for αvβ8 integrincompared to the mouse hybridoma antibody ADWA11 and some Fabs havingsingle amino acid substitutions retained binding for hαvβ8.

Example 7: Evaluation of Anti-αvβ8 Integrin Antibodies by Biacore™Method

Biotinylated recombinant αvβ8 integrin was captured on astreptavidin-coated Biacore™ chip (GE Healthcare Life Sciences) and thebinding response versus time for Fab fragments was measured over aseries of Fab concentrations. Representative background subtractedBiacore™ sensograms overlaid with the kinetic curve fits were obtained.

More specifically, recombinant αvβ8 integrin (e.g., R&D Systems) wasbiotin labeled via primary amines and immobilized on a Sensor Chip SAusing a Biacore™ T200 instrument (GE Healthcare Life Sciences). Fabbinding experiments were performed at 25° C. using a 30 μl/min flow ratein 0.01 M HEPES pH 7.4, 0.15 M NaCl, 1-2 mM MgCl₂, and 0.005% v/vsurfactant P20 (HBS-P) buffer. Association was monitored for 5-10minutes and dissociation for a further 15-20 minutes for each Fabconcentration. After each injection, the chip surface was regeneratedwith IgG elution buffer (Thermo Fisher Scientific). All data wasanalyzed using the Biacore™ T200 Evaluation software. Data was dualbackground subtracted using the adjacent flow cell coupled withstreptavidin without captured integrin, and buffer only injections.Kinetic constants for at least three experiments were obtained andreported as the mean.

Results

Antibody biding to αvβ8 integrin from various species was evaluated. Themouse hybridoma ADWA-11 Fab and the humanized ADWA-11 VH05-2/VK01(2.4)Fab were both found to cross react with the αvβ8 integrin of otherspecies. The mouse hybridoma ADWA-11 Fab cross reacted with human αvβ8integrin, cynomolgus monkey αvβ8 integrin, and mouse αvβ8 integrin. Thehumanized ADWA-11 VH05-2/VK01(2.4) Fab cross reacted with human αvβ8integrin, cynomolgus monkey αvβ8 integrin, and mouse αvβ8 integrin.Additionally, these antibodies were evaluated for their ability to bindto related integrins hαvβ3 or hαvβ6. These antibodies were specific forhαvβ8 and did not bind to the closely related integrins hαvβ3 or hαvβ6in a Biacore assay, as shown in FIG. 5.

A comparison of the affinity measurements listed in Table 6 shows thatthe humanized ADWA-11 Fab had a lower KD for binding to both human αvβ8integrin and mouse αvβ8 integrin, as compared to the KD of the mousehybridoma ADWA-11 Fab for binding to human αvβ8 integrin and mouse αvβ8integrin. More specifically, the humanized ADWA-11 Fab had about a2.5-fold lower KD for binding to human αvβ8 integrin and about a3.5-fold lower KD for binding to mouse αvβ8 integrin, as compared to therespective KD values of the mouse hybridoma ADWA-11 Fab. These data showan overall substantial affinity improvement for the humanized anti-αvβ8antibody over the mouse hybridoma antibody (FIG. 6A-6B and Table 6).

To evaluate the monomeric KD of the Fab ADWA11 2.4, also referred toherein as ADWA-11 VH05-2/VK01(2.4) and ADWA11 5-2 2.4, and the parentalmouse IgG, recombinant human, cynomolgus, mouse, and rat αvβ8 wasimmobilized on a Biacore chip and the ka and kd of the Fabs weredetermined as generally described herein (Table 6). ADWA11 2.4demonstrated an equivalent affinity for human, cynomolgus, mouse, andrat αvβ8 with a KD of <200 pM, however due to the very slow kd precisedetermination of KD was not possible. The parental mouse IgGdemonstrated an equivalent affinity KD for human, cynomolgus, and mouseαvβ8 (KD of 489-536 pM) (rat was not tested) (FIG. 6C).

Additional Biacore experiments refined the estimated KD values forADWA11 2.4 to a KD of <100 pM for human and cynomolgus αvβ8 and70.8±19.9 pM (Average±Standard Deviation) for mouse αVβ8.

TABLE 6 αvβ8 Integrin species affinity as assessed by Biacore (n > 3)Fab αvβ8 species ka (l/Ms) kd (l/s) KD Mouse hybridoma antibody ADWA-11human 9.22E + 04 4.86E − 05 5.36E − 7M Also referred to herein as: mouse1.03E + 05 5.03E − 05 4.89E − 7M Mouse ADWA11 cyno 9.81E + 04 4.97E − 055.07E − 7M ADWA11 5-2 2.4 human 1.61E + 05 <3E − 05 <2E − 010 Alsoreferred to as: cyno 1.72E + 05 <2E − 05 <2E − 010 ADWA11 2.4 mouse2.57E + 05 1.82E − 05 <2E − 010 VH05-2_VK01(2.4) rat 3.41E + 05 <2E − 05<2E − 010 Humanized ADWA11 human 1.73E + 05 3.61E − 05 2.09E − 7M ADWA11VH05-2_VK01 mouse 1.77E + 05 2.43E − 05 1.37E − 7M VH: SEQ ID NO: 39human 2.00E + 05 2.37E − 05 1.18E −10M VL: SEQ ID NO: 47 mouse 2.20E +05 <2E − 5 <1E − 10M Also referred to as: rat 1.94E + 05 3.10E − 051.60E − 10M ADWA11 VH05/VK01 cyno 2.25E + 05 <2E − 5 <1E − 10MVH05-2_VK01; VH05-2_VK01 parental; and VH05/VK01 Fab

To evaluate the specificity of ADWA11 2.4, recombinant human αvβ6 andαvβ3 were immobilized on a Biacore chip and binding of the parentalmurine and humanized mAb variant of ADWA11 2.4 was determined. Theparental hybridoma and humanized mAb did not bind αvβ3 or αvβ6, whilebinding to αvβ8 in separate BIAcore experiments was observed. Apan-Integrin αV antibody was used to demonstrate immobilization of αvβ3or αvβ6 recombinant protein on the Biacore chip. Thus, this example alsodemonstrates the specificity of the humanized antibody for αvβ8.

Example 8: Evaluation of Anti-αvβ8 Integrin Antibodies in Cell BindingAssays Methods

Cell binding experiments were performed with human glioblastoma U251(Sigma) or C8-S (ATCC) cells cultured in MEM 10% hiFBS. The cells grownto 70-90% confluence were detached with 0.05% trypsin and washed twotimes with PBS containing 2% BSA.

For cell-binding experiments with HEK293-F cells overexpressing humanαvβ3, αvβ5, αvβ6, αvβ8. HEK293-F cells transiently expressing fulllength human integrin beta 3 (Accession No. NP 000203.2), human integrinbeta 5 (Accession No. NP 002204.2), human integrin beta 6 (Accession No.NP 000879.2), or human integrin beta 8 (Accession No. NP 002205.1) wereprepared using proprietary vectors and vendor provided protocols. Cellswere harvested after 4 days and analyzed for integrin expression. Tocharacterize Integrin αvβ3, αvβ5, αvβ6, αvβ8 expression 100,000 cellswere incubated in the indicated commercially available or directlyconjugated proprietary antibodies for 30 minutes, along with LIVE/DEADfixable cell stain (Invitrogen) at 1:2000 to distinguish live cells.Cells were spun down for five minutes at 300 g force. The cells werethen washed twice with wash buffer (PBS+0.2% BSA) to remove excessunbound antibodies and analysed on BD biosciences Fortessa flowcytometer

Live cell-binding protocol: 100,000 cells were incubated with a dilutionseries of the anti-αvβ8 or human IgG1_3mut Isotype antibody for three tofour hours on ice. The cells were spun down for five minutes at 300 gforce. The cells were then washed three times with wash buffer (PBS+0.2%BSA) to remove excess unbound antibodies. After thorough washing, cellswere incubated with Fab′2 anti-human Fc-PE (Invitrogen) or goat Fab′2anti-mouse-APC (Jackson Labs) at 1:1000 dilution along with LIVE/DEADfixable cell stain (Invitrogen) at 1:2000 to distinguish live cells. Thewhole content was incubated for 30 minutes on ice. After washing, thestained cells were analyzed on BD biosciences Fortessa flow cytometer(gated for live cells) using FlowJo analysis software. The meanfluorescence intensities (MFI) at different antibody concentrations wereplotted for various antibodies.

Fixed cell-binding protocol: In some instances, cell binding experimentswere performed with human glioblastoma U251 (Sigma) cells cultured inMEM or 10% hiFBS. The cells grown to 70-90% confluence were detachedwith 0.05% trypsin and washed two times with PBS containing 2% BSA. U251(25,000 cells) were incubated with LIVE/DEAD fixable cell stain(Invitrogen) at 1:2000 to distinguish live cells. The cells were spundown for 5 minutes at 300 g force, and washed with wash buffer (PBS+0.2%BSA), followed by fixation with 2% paraformaldehyde for 10 minutes atroom temperature. Fixed cells were wasted twice with wash buffer,followed by addition of a dilution series of anti-αvβ8 antibody ofinterest for 1 hour at 37 degrees. The cells were washed three timeswith wash buffer to remove excess unbound antibodies. After throughwashing, Fab′2 anti-human Fc-PE (Invitrogen) or anti-human Kappa LightChain-APC (Invitrogen) detection antibody at 1:1000 dilutions was addedto cells and incubated for 30 minutes on ice. After washing, the stainedcells were analyzed on BD biosciences Fortessa flow cytometer (gated forlive cells) using FlowJo analysis software. The mean fluorescenceintensities (MFI) at different antibody concentrations were plotted forvarious antibodies.

Results

Fixed U251 cell binding data for ADWA11 VH05/VK01 Fabs having singleamino acid substitutions in either the heavy chain variable region,including F64V, or the light chain variable region, including L305,M94Q, L97Y, F101L, F101W, or Q105G, or a combination of amino acidsubstitutions referred to as 2.1, 2.2, 2.3, 2.4, 2.1 (F64V), 2.2 (F64V),2.3 (F64V), and 2.4 (F64V) in Table 5 were obtained and compared to theparental antibody (FIGS. 7A-7C).

U251 cell binding data was also obtained for the antibodies mIgG_4mutand hIgG_VH05VK01 (also referred to as ADWA11 2.4 and described furtherin Example 5)(FIG. 8). The apparent affinity of mIgG_4mut andhIgG_3mut_VH05VK01, for U251 cells is shown below in Table 7. These datademonstrate that hIgG_3mut_VH05VK01 was determined to have a higheraffinity as compared to mIgG_4mut.

TABLE 7 Affinity values from U251 cell binding assay ADWA11 antibodyU251 app Kd (pM) mlgG_4mut 823 hlgG 3mut VH05VK01 430

The apparent-affinity of ADWA11 2.4 for U251 (human glioblastoma) orC8-S (mouse astrocyte) cells was also evaluated in a cell-binding assay,according to the methods as generally described herein. Briefly, cellswere incubated with a serial dilution of ADWA11 2.4 for 4 hours on ice,followed by detection on bound antibody with an anti-human-PE secondaryantibody, and analyzed by flow cytometry. ADWA11 2.4 demonstratedsaturable binding to human αvβ8 and mouse αvβ8-expressing cells, with anaverage EC50 in the U251 (human αvβ8) cell-binding assay of 126 pm witha standard deviation of plus or minus 34 pM (FIG. 9A; n=3).

In further studies, the EC50 of ADWA11 VH05-2/VK01(2.4) binding to humanU251 cells was determined to be 256±115 pM (average±standard deviationin seven independent experiments) and binding to mouse C8-S cells wasdetermined to be 145±23.7 pM (average±standard deviation in fourindependent experiments).

HEK293F cell-binding experiments with transiently overexpressed integrinfamily members demonstrate ADWA11 2.4 specifically binds to cellsexpressing human αvβ8, but not αvβ3, αvβ5, or αvβ6 (FIG. 9B). Thus,ADWA11 2.4 demonstrated improved characteristics compared with theparent mouse antibody ADWA11 thereby suggesting that the humanizedADWA11 2.4 is a potential improved human therapeutic.

Example 9: Evaluation of Anti-αvβ8 Integrin Antibodies in a αvβ8 InducedTGF-β Activation Assays Methods

The effect of anti-αvβ8 antibodies on TGF-β pathway trans-activation wasmeasured using U251-MG (Sigma) and Mv1Lu-SMAD-luciferase reporter cells.In some experiments, C8-S mouse astrocyte cells were used instead ofU251 cells. Briefly, mink lung epithelial cell line MvLu1 cells (ATCC)were transduced with Cignal SMAD reporter (luc) lentiviral particles(SABioscience) at a multiplicity of infection (MOI) of 50. Stable celllines expressing the SMAD firefly luciferase construct were generated byculturing the cells in the complete growth media (MEM plus 10% fetalbovine serum (FBS) with L-glutamine+penicillin/streptomycin)supplemented with 2 μg/mL puromycin. For the experiment, U251 cells(5000 cells in 50 μL in MEM medium containing 2% charcoal-stripped FBS)were added to each well of a clear-bottom, white-walled TC-treated 96well plate and incubated for 1 hr at 37° C. A dilution series ofanti-αvβ8 antibodies, e.g., FAB antibodies, was prepared in MEM mediumcontaining 2% charcoal-stripped FBS and added to the plated U251 cells,25 μl per well. After an hour of incubation, Mv1Lu-SMAD-luciferasereporter cells were added (5000 cells/well in 25 μL) to each well andafter 18 hours of incubation at 37° C. the luciferase activity wasmeasured using Bright Glo reagent (Promega) according to manufacturessuggested protocol. Luminescence was measured using an Envision platereader with 1 s integration time.

Results

Inhibitory activity of various antibodies on αvβ8 induced TGF-βtransactivation of U251 cells was monitored by assessing decreasedluciferase activity (FIG. 10A-10F).

FIG. 10A depicts a comparison of antibodies generated as described inExample 3, and include a comparison between ADWA11 VH05/VK01 (alsoreferred to as VH05-VK01 Fab) to the parental mouse hybridoma antibodyADWA11 (mFab). The EC50 value measured by apparent affinity in the TGF-βtransactivation assay was improved from 4.6 nM for the parental mousehybridoma antibody ADWA11 (mFab) to 1.3 nM for ADWA11 VH05/VK01 (alsoreferred to as VH05-VK01 Fab).

The impact of amino acid substitutions made in either the heavy chainvariable region, or the light chain variable region of ADWA11 VH05/VK01on TGFβ transactivation by U251 cells was also assessed (FIG. 10B-10D).Fabs having a F101L or F101W single amino acid substitution in the lightchain variable region displayed a reduced TGFβ transactivation, ascompared to the ADWA11 VH05/VK01 Fab.

A comparison of the effect of humanized ADWA-11 IgG molecules on αvβ8induced TGF-β transactivation of U251 cells was also monitored using theluciferase activity assay (FIG. 10E). These data show that theVH05/VK01-D61E and -N55Q-D61E mutants, have comparable effects on TGFβtransactivation.

To evaluate the potency of ADWA11_VH05-2_VK01(2.4), alternativelyreferred to herein as ADWA11 2.4, a co-culture system was establishedwith human and mouse cells that endogenously express αvβ8 with aTGFβ-sensitive luciferase reporter cell system. Briefly, U251 (humanglioblastoma) or C8-S (mouse astrocyte) cells were plated with mink lungepithelial cell (Mv1Lu) stably transduced with Cignal SMAD reporter(luc) lentiviral particles (SABioscience) at a multiplicity of infection(MOI) of 50 (Mv1Lu-Smad cells). Mv1Lu-Smad cells respond to TGFβgenerated by the U251 or C8-S cells, and inhibition of αvβ8 function canbe monitored by a decrease in luciferase activity. FIG. 10F shows theeffect of ADWA11 2.4 on TGFβ transactivation by U251 cells and C8-S,compared to an isotype negative control antibody.

These data demonstrate that ADWA11 2.4 is a more potent inhibitor ofαvβ8 induced TGF-β transactivation than other antibodies, including themouse ADWA11 monoclonal antibody. The IC50 for ADWA11 VH05-2/VK01(2.4)in the TGFβ transactivation assay with U251 cells was determined to be199±93.6 pM (Average±Standard Deviation in five independentexperiments).

Example 10: Evaluation of the Immunogenicity of Anti-αvβ8 IntegrinAntibodies

The functional significance of peptides binding to MajorHistocompatiblity Complex (MHC) was evaluated by a T cell proliferationassay. CD4, a transmembrane glycoprotein expressed on T-helper cells,recognizes peptides bound MHC Class II molecules on the surface ofantigen presenting cells (APC). This interaction results inproliferation of T-helper cells leading to an immune response. T-Cellproliferation was monitored by a decrease in the fluorescence intensityof the individual cells containing carboxyfluorescein succinimidyl ester(CFSE) dye. ProImmune's REVEAL® Immunogenicity System T cell assay usesflow cytometry methods to analyze division of CFSE dye labelled cells.PMBCs from various donors were incubated with CFSE to form intracellularfluorescent conjugates. Fluorescence intensity of CFSE is halved througheach consecutive cell division, thus allowing measurement of cellproliferation. This reliable and reproducible CFSE-labelled T cell assayis useful to determine potential CD4-Tcell epitopes on MHCII presentedpeptides. Peptide controls derived from Influenza/Tetanus and TuberculinPurified Protein Derivative (PPD) were used as positive controls forcell proliferation.

Twenty-nine peptides encompassing the CDRs were tested with PBMC derivedfrom 51 donors for T-cells proliferation (Table 1, SEQ ID NOs: 94-122).The CFSE labelled PBMCs were incubated with test peptides for seven daysand the extent of CD4+ T cells proliferation was monitored byFlow-cytometry method. The number of responders for the peptide derivedfrom optimized molecule was compared with the number of responders forcorresponding germline sequence peptides.

Reference antigens comprising known MHC class II epitopes were used inthis study Tuberculin Purified Protein Derivative (PPD) is a derivativeof Mycobacterium tuberculosis, and was used at a final assayconcentration of 5 μg/ml. Approximately 70-100% of the PBMC donors areexpected to react to this protein as a result of previous vaccination,i.e., through a memory immune response.

Keyhole Limpet Hemocyanin (KLH) is a recognized and potent naïve proteinimmunogen, used at a final concentration of 0.25 mg/ml in the assay.Typically between 50-80% of donors might be expected to react to thisprotein, presumably driven by a naïve immune response.

The synthetic peptide HA is derived from Influenza A hemagglutinin(PKYVKQNTLKLAT, residues 307-319; SEQ ID NO: 129) and was used at afinal assay concentration of 5 μM. It is expected to elicit a responsein up to 50% of donors. The synthetic peptide TT (AQYIKANSKFIGITEL (SEQID NO: 130), TET 830 modified/T-helper epitope from tetanus toxoid) is auniversal human tetanus toxin T cell epitope that induces T-cellactivation and is used as a helper peptide in vaccinations. It was usedat a final assay concentration of 5 uM, with up to 45% of donorsexpected to respond.

The F64V substitution (SEQ ID NO: 105) in the heavy chain was found toincrease the immunogenicity. Eleven of 51 donors' PBMCs responded in theCD4+T cells proliferation assay and none of the donors were sensitive tothe corresponding germline sequence. Other substitutions including Q105G(SEQ ID NO: 111), L30S (SEQ ID NO: 106), M94Q (SEQ ID NO: 107), and N58S(SEQ ID NO: 113) were found to reduce the immunogenicity each to adifferent extent as shown in Table 8. Percentage responder for differentCDR peptides compared to positive control peptides are given in FIG. 11.

TABLE 8 Number of responders* for peptides corresponding to differentsubstitution After substitution Original substitution F64V 0 11 Q105G 63 L30S 6 0 M94Q 6 0 N58S 5 3 *numbers of donors PBMC were responding inthe CD4+ T cells proliferation assay

These data demonstrate that certain antibodies of the invention,including ADWA11 2.4, exhibit decreased T cell responses compared withthe mouse parent mAb ADWA11. These results suggest that ADWA11 2.4 is animproved potential human therapeutic compared to its mouse parentantibody.

Example 11: Inhibition of αvβ8 Improves the Efficacy of Anti-PD-1Therapy in the EMT6 Tumor Model Methods

In this study, 3×10⁵ EMT6 (ATCC) cells were implanted into the fourthmammary fat pad or subcutaneously in Balb/c mice (Charles RiverLaboratory). Mice were randomized into treatment groups when theirtumors reached an average of 50 mm³ and then treatment was initiated.For treatment, mice received a dose of 10 mg/kg of the indicatedantibodies 2A3_rat IgG (“2A3”; BioXcell), anti-PD-1 antibody (“PD1”;clone RMP1-14, BioXcell), 2B8_mIgG1_4mut (“2B8”), or ADWA11_mIgG1_4mut(ADWA11) by intravenous injection every four days for a total of threedosage administrations. Tumors were measured in two dimensions tomonitor growth, where volume (V)=½ L×W², and L (length) is defined asthe longest diameter of the tumor and W (width) is perpendicular to L.Tumor measurements were recorded 2-3 times per week until end of thestudy.

Results

Anti-αvβ8 (ADWA11) in combination with anti-PD1 therapy (ADA11+PD1)synergistically and significantly decreased tumor growth and improvedsurvival over anti-PD1 or anti-αvβ8 monotherapy treatment groups (FIGS.12A and 12B).

In the 10 mg/kg monotherapy dose group ADWA11 treatment resulted in a47.1% tumor growth inhibition (TGI) on Day 13 of the study however, theTGI was transient and no mice reached the end of the study (0% survivalat Day 51). Anti-PD1 monotherapy treatment resulted in a 15% TGI on Day14 of the study and no mice reached the end of the study (0% survival atDay 51). By comparison, ADWA11 (10 mg/kg dose group) in combination withanti-PD-1 antibody resulted in a 90.0% TGI on Day 14 of the study and a60% of mice reached the end of the study (60% survival at Day 51).

These results demonstrate for the first time that ADWA11 antibodies,including ADWA11 2.4, provide a synergistic therapeutic effect whencombined with an inhibitor of PD-1, e.g., anti-PD-1 antibody. These datasuggest that ADWA11 2.4 is a potential human therapeutic that canprovide a synergistic therapeutic anti-tumor response when combined witha PD-1 inhibitor.

Example 12: Inhibition of αvβ8 Improves the Efficacy of 4-1BB andAnti-CTLA4 Therapy in the EMT6 Model Methods

In this EMT6 tumor efficacy study, 1×10⁶ EMT6 (ATCC) cells wereimplanted into the fourth mammary fat pad in Balb/c mice (Charles RiverLaboratory). Mice were randomized when tumors reached an average of 100mm³ and treatment was initiated. Mice received i.v. dosing of 4-1BB(MAB9371 R&D Systems, 1 mg/kg Day 0 and Day 4), anti-CTLA4 (clone 9D9BioXcell, 10 mg/kg Day 0, Day 4, and Day 8), anti-αvβ8 (ADWA11, 10 mg/kgDay 0, Day 4, and Day 8), 2B8_mIgG_4mut (10 mg/kg, Day 0, Day 4, and Day8), or 2A3_rat IgG (BioXcell, 10 mg/kg Day 0, Day 4, and Day 8). Tumorswere measured in two dimensions to monitor growth, where volume (V)=½L×W², and L (length) is defined as the longest diameter of the tumor andW (width) is perpendicular to L. Tumor measurements were recorded 2-3times per week until end of the study.

Immunohistochemistry (IHC) analysis: 4 mm thickness formalin fixedparaffin embedded (FFPE) tumor tissue sections for CD8, CD45, andGranzyme B expression using custom protocols and Leica Bond-maxautomated IHC stainer. Images were acquired on a Leica/Aperio AT2 wholeslide digital scanner using the 20× magnification setting. Images wereanalyzed using custom algorithms created in Visiopharm 7.2 software andoptimized for each target of interest. Cell counting was carried out onthe viable tissue and was normalized by the viable tissue area. CellDensity was calculated using the following equation:Cells/μm2=(#Positive Cells/Viable Tumor area (μm2))*1×106. Percentobject density was calculated using the following equation: Area ofstaining (μm2)/Viable Tumor Area (μm2).

Results

Anti-αvβ8 (ADWA11) in combination with anti-4-1BB or anti-CTLA4significantly and synergistically decreased tumor growth and improvedsurvival over anti-4-1BB, anti-CTLA4, or anti-αvβ8 monotherapy treatmentgroups (FIG. 13).

Changes in tumor infiltrating cell density after treatment withanti-αvβ8 antibody were assessed (FIG. 15). Lymphocyte abundance wasquantified in the EMT6 tumor model by IHC analysis of the density ofCD45 (total lymphocytes and myeloid cells), CD3 (total T cells), CD4 Tcells, CD8 T cells, and Granzyme B (activated CD8 and NK cells)staining. These data demonstrated that anti-αvβ8 monotherapy increasedthe abundance of the total CD45+ cells, CD4+ T cell, and CD8+ T cells,and resulted in a very significant increase in the density of Granzyme Bexpressing cells (n=10 for each group).

Tumor lymphocyte abundance in tumor tissue was analyzed Day 11 (antibodytreatment on Day 0, 3, 6, 9) from mice treated with Isotype control orADWA11 (2.4) (FIG. 15). ADWA11 (2.4) treatment increased the density oftotal leukocytes (CD45+, 1540±558 vs 2470±407), CD8 T cell (CD8+,76.3±62.7 vs 170±74.2) and cytotoxic cells (% Granzyme B density,11.8±11.0 vs 106±35.1) in the tumor microenvironment (average number ofcells CD45+, average number of cells CD8+, or average % Granzyme Bstaining area per mm²±standard deviation in Isotype vs ADWA11 (2.4)treatment)

These results demonstrate for the first time that ADWA11 antibodies,including ADWA11 2.4, provide a synergistic therapeutic effect whencombined with an agonist of 4-1BB, e.g., anti-4-1BB antibody. These datasuggest that ADWA11 2.4 is a potential human therapeutic that canprovide a synergistic therapeutic anti-tumor response when combined withan agonist of 4-1BB.

These results also demonstrate for the first time that ADWA11antibodies, including ADWA11 2.4, provide a synergistic therapeuticeffect when combined with an inhibitor of CTLA4, e.g., anti-CTLA4antibody. These data suggest that ADWA11 2.4 is a potential humantherapeutic that can provide a synergistic therapeutic anti-tumorresponse when combined with an inhibitor of CTLA4.

Example 13: Inhibition of αvβ8 Improves the Efficacy of RadiationTherapy in the CT26 Tumor Model Methods

CT26 tumor efficacy study: 4×10⁵ CT26 (ATCC) cells were implantedsubcutaneously into the flank of Balb/c mice (Charles River Laboratory).Mice were randomized when tumors reached an average of 100 mm³ andtreatment was initiated. Mice received 10 mg/kg i.v. dose every 4 days,3 total doses, of 2B8_mIgG1_4mut (in house) isotype control orADWA11_mIgG1_4mut, and a single dose of 5Gy tumor targeted radiation onDay 5 after first dose. Tumors were measured in two dimensions tomonitor growth, where volume (V)=½ L×W², and L (length) is defined asthe longest diameter of the tumor and W (width) is perpendicular to L.Tumor measurements were recorded 2-3 times per week until end of thestudy.

qPCR analysis of gene expression: tumor tissue was collected and 30 mgof tissue was homogenized in 900 μL of lysis buffer supplied in theRNeasy Plus Mini Kit, using Omin Bead Ruptor. RNA from homogenized tumorsamples was isolated using the RNeasy Plus Mini Kit and vendorrecommended protocols. cDNA was synthesized using 2 μg of total RNA andthe High-capacity cDNA reverse transcription kit, using vendorrecommended protocols. Gene expression was analyzed using 50 ng of cDNAand gene-specific taqman primers, TaqMan Universal Master Mix II, andvendor recommended protocols. ViiA7 real-time qPCR system was used forqPCR studies. The threshold cycles (CT) for each sample was analyzedusing the recommended comparative CT method and expression of targetgenes is reported as fold change of treatment group compared to isotypecontrol group. A two-tailed unpaired Students T-test test was used tocompare treatment group to the isotype control group with significancereported at <0.05.

Immunohistochemistry (IHC) analysis: 4 mm thickness formalin fixedparaffin embedded (FFPE) tumor tissue sections were prepared for CD8,CD45, and Granzyme B expression using custom protocols and LeicaBond-max automated IHC stainer. Images were acquired on a Leica/AperioAT2 whole slide digital scanner using the 20× magnification setting.Images were analyzed using custom algorithms created in Visiopharm 7.2software and optimized for each target of interest. Cell counting wascarried out on the viable tissue and was normalized by the viable tissuearea. Cell Density was calculated using the following equation:Cells/μm2=(#Positive Cells/Viable Tumor area (μm2))*1×106.

Results

Anti-αvβ8 antibody (ADWA11) in combination with radiation therapysignificantly decreased tumor growth and improved survival overradiation therapy alone (FIG. 14A).

In the CT26 studies, ADWA11 antibody (anti-ITGαVβ8) monotherapy resultedin a 64.3% TGI on Day 19 of the study, but the response was transientand only 1 out of 10 mice reached the end of the study (10% survival,Day 57). Radiation therapy alone (5 Gray (Gy) dose on Day 5) resulted ina 57.7% tumor growth inhibition (Day 18), and 1 out of 20 mice reachedthe end of the study (5% survival, Day 57). By comparison ADWA11treatment in combination with radiation therapy resulted in an 87.7%tumor growth inhibition (Day 19, 10 mg/kg ADWA11 and 5 Gy radiationtherapy dose group) and 9 out of 19 mice reached the end of the study(47.4% survival, Day 57).

To investigate the effect of ADWA11 on tumor lymphocyte abundance in theCT26 tumor model, tumor tissue was collected Day 12 (antibody treatmenton Day 0, 4, 8) from mice treated with Isotype control or ADWA11VH05-2/VK01 (anti-ITGαVβ8) and analyzed for lymphocyte markers by IHC(FIG. 14B, top panel). ADWA11 VH05-2/VK01 treatment increased thedensity of total CD45+ leukocytes; Isotype: 367±128, ADWA11 VH05-2/VK01:695±94.8 (Average number of cells per mm²±standard deviation). CD8+ Tcell; Isotype: 173±79.3, ADWA11 VH05-2/VK01: 374±80.4 (Average number ofcells per mm2±standard deviation). Ganzyme B expressing cytotoxic cells,Isotype: 264±65.5, ADWA11 VH05-2/VK01: 514±91.7 (Average number of cellsper mm2±standard deviation) in the tumor microenvironment. Additionally,anti-ADWA11 treatment in combination with radiation therapy increasedthe mRNA expression level of CD45 (3.86±0.979), CD8a (5.45±3.53),Granzyme B (4.21±1.02), and IFNγ (5.53±2.13) in the tumormicroenvironment (Fold Change±standard deviation vs Isotype treatmentgroup) (FIG. 14B, bottom panel).

These studies demonstrate that anti-ITGαVβ8 (ADWA11_mIgG1_4mut)treatment increases the abundance of activated lymphocytes in the tumormicroenvironment, and in combination with tumor targeted radiation isefficacious at causing tumor regression and long-term survival.

These results also demonstrate for the first time that ADWA11antibodies, including ADWA11 2.4, provide a synergistic therapeuticeffect when combined with radiation therapy. These data suggest thatADWA11 2.4 is a potential human therapeutic that can provide asynergistic therapeutic anti-tumor response when combined with radiationtherapy.

Example 14: Evaluation of Integrin αvβ8 as a Novel Suppressor of TumorImmunity and Target for Tumor Immunotherapy

In this study, a potent αvβ8 blocking monoclonal antibody (ADWA-11) thatwas generated by immunizing Itgb8 knockout mice with recombinant αvβ8,was used to examine whether inhibition of this integrin could facilitateanti-tumor immunity.

In non-neoplastic tissues, αvβ8 integrin is expressed on neuroepithelia,fibroblasts, dendritic cells, and T cells, and can activate latenttransforming growth factor β (TGFβ), an important immunomodulator. Asnow shown herein, within carcinomas, myeloid cells express αvβ8 integrinand that a potent monoclonal antibody blocking αvβ8 (ADWA-11) causesgrowth suppression or complete tumor regression in syngeneic models ofsquamous cell carcinoma, breast and colon cancer, especially whencombined with other immunomodulators (anti-PD1, anti-CTLA-4 or 4-1BB) orradiotherapy. Treatment with ADWA-11 increases tumor infiltration andGranzyme B expression of CD8+ T cells, and enhances the ratio ofpro-inflammatory to suppressive tumor associated macrophages. Most humantumors express ITGB8 mRNA and, as shown herein, there are high levels ofsurface αvβ8 expression on monocytes, macrophages, and dendritic cellsubsets in biopsies of human ovarian and renal cell carcinomas. Thesefindings identify αvβ8 integrin as a promising new target for cancerimmunotherapy.

Efficacious Synergistic Combination Immunotherapy with ADWA-11 andAnti-PD-1 in a Squamous Cell Carcinoma Model

The effects of ADWA11 alone, or in combination with an anti-PD-1antibody, were examined in a syngeneic tumor model of establishedsquamous cell carcinoma (CCK168 cells) (FIG. 16A and FIG. 28). CCK168cells, a chemically induced squamous cell carcinoma cell line derivedfrom FVB mice, were injected subcutaneously into the dorsolateral rightflank of syngeneic wild type FVB mice at a dosage of 1.5×10⁴cells/mouse.

Tumors were allowed to grow over 14 days. Mice selected for theexperiment had tumor size of at least 5 mm in diameter and wereoptimally distributed based on tumor sizes, between different treatmentgroups using studylog software. Mice were weighed and tumor size wasmeasured every other day for the duration of the study using a traceabledigital caliper (Fisher Scientific, model #14-648-17). Mice wereeuthanized when tumor size reached or exceeded 2000 mm³ or developed alarge ulceration at the tumor site.

Mice were treated with hybridoma antibody ADWA11 or isotype-matchedcontrol antibodies on days 0 and 7 and with mouse anti-PD-1 antibody(RMP1-14, BioXcell) or its isotype-matched control antibody on days 0, 4and 8 (day 0 being the first day of therapy). Appropriate antibody foreach group and isotype control antibodies were injectedintraperitoneally at doses of 10 mg/Kg for each antibody, ADWA11,anti-PD-1 antibody (RMP1-14, BioXcell), control antibody ADWA-21 (forADWA11), and control 2A3 (BioXcell). Control ADWA-21 binds human but notmouse integrin-β8.

CCK168 tumors demonstrated minimal responses to an anti-PD1 antibody,but five of ten mice treated with ADWA11 monotherapy showed tumorregression (FIG. 16B-16C). Combination therapy with ADWA11 and ananti-PD1 antibody induced complete regression of eight out of tentumors, and significant increase in overall survival when mice weretreated with hybridoma ADWA11 and an anti-PD1 antibody (FIG. 16B) orADWA11_4mut_mIgG1 and anti-PD1 antibody (FIG. 28).

Surviving mice were observed for up to two years after tumor regressionand none showed evidence of subsequent tumor regrowth. Thirteen micefrom two replicate experiments with complete regression aftercombination therapy and 3 mice with complete regression aftermonotherapy with ADWA11 were re-challenged either once or twice withCCK168 cells and there was no tumor growth in any, demonstrating thedevelopment of long-term tumor immunity.

These results demonstrate for the first time that ADWA11 antibodies,including ADWA11 2.4, provide a synergistic therapeutic effect whencombined with an inhibitor of PD-1, e.g., an anti-PD-1 antibody. Thesedata suggest that ADWA11 2.4 is a potential human therapeutic that canprovide a synergistic therapeutic anti-tumor response when combined witha PD-1 inhibitor.

Surface integrin αvβ8 is present on myeloid cells within the tumormicroenvironment

Flow cytometry was utilized to identify which cell types of the tumorexpress integrin β8, presumed to be expressed as an αvβ8 heterodimer(FIG. 17A and FIG. 17B). αvβ8 expression was readily detectable ongreater than 80% of CD45+CD11b+F4/80+CD64+ macrophages and less than 20%of CD45+CD11b+F4/80-CD64-CD11c+MHCII^(high) dendritic cells withinCCK168 tumors (FIG. 17B). αvβ8 was expressed at similar levels in eachof the macrophage subpopulations examined, including early infiltrating(pro-inflammatory) Ly6C+ cells and immunosuppressive CD206+ cells (FIG.17B). αvβ8 was also expressed on CCK168 tumor cells in vitro (FIG. 18B).Low levels of expression of αvβ8 on CD45-tumor and stromal cells in vivowas also found (FIG. 18A). No expression of αvβ8 was found onintratumoral T cells (FIG. 20).

Animals

All animal studies were performed in accordance with approved protocolsby the University of California, San Francisco, Institutional AnimalCare and Use Committee of Pfizer, Inc. Institutional Animal Care and UseCommittee. Wild type FVB/N mice used were either purchased from JacksonLaboratories (The Jackson Laboratories, stock #001800) or derived fromour own breeding colony derived from this stock. Wild type Balb/c micewere purchased from Charles River Laboratories (Charles RiverLaboratories, strain code 028).

Human Tumor Processing

For all human samples, informed consent was obtained from all subjects,and work was performed in accordance with Institutional Review Board(IRB) approval. Fresh tissue was collected and processed through theUCSF Immunoprofiler workflow, a translational platform developed andoptimized for profiling immune subsets within cancer. Briefly, tissuewas acquired from the operating room and transported to the laboratorywithin 4 hours of excision. Tissue was vigorously minced (<1 mm chunks)and digested enzymatically (3 mg/ml collagenase A, 50 U/ml DNase I)based on developed standard operating procedures. Immune populationswere subjected to multiplexed flow cytometry (>60 colors) to analyzeproportionality and mean fluorescent intensities of known subsets andtheir expression of αvβ8. All antibodies were purchased from BDPharmingen, eBioscience, Invitrogen, or BioLegend. Anti-αvβ8 antibodywas generated as described previously. All flow cytometry including cellsorting was performed on a special-order BD FACSAria Fusion flowcytometer. Analysis of flow cytometry data was done using FlowJo.

Flow Cytometry Protocol

Subcutaneous tumors were isolated from the mice using scissors and bluntdissection. The tumors were placed in a petri dish with digestioncocktail of Collagenase XI (Sigma C9407) 2 mg/mL, Hyaluronidase (SigmaH3506) 0.5 mg/ML, and DNase (Sigma DN25) 0.1 mg/mL prepared in C10 media(RPMI 1640, Hepes 1%, Penicillin/Streptomycin 1×, fetal calf serum 10%,sodium pyruvate 1 mM, non-essential amino acids 1×, andbeta-mercaptoethanol 0.45%). Tumors were minced using sterile scissors.The resultant slurry of cells were transferred into 50 mL conical tubes(Fisher Scientific #14-432-22) and the petri dish used to mince thetumor was rinsed with 2 mL C10 media to capture remaining cells. Cellswere incubated in a shaker at 255 rpm for 45 minutes at 37° C. Afterincubation, 15 mL of C10 media was added to the digested tumor cells andgently vortexed for 15 seconds. The cell slurry was passed through a 100μm mesh strainer (Falcon® #352360) into a clean 50 mL conical tube.Cells were pelleted by centrifugation for 5 minutes at 200 g at 4° C.and reconstituted in PBS. Cell counts were performed using a CountlessII FL hemocytometer (Life Technologies).

Isolated single cell preps were used for cell surface and intracellularstaining. After counting, 10×10⁶ cells were transferred into each wellof a v-shaped 96-well plate for staining. Live dead staining with GhostDye™ Violet 510 (TONBO bioscience #13-0870) at 1:1000 for 20 minutes at4° C. Fc receptor and non-specific binding was blocked with anti-CD16/30(eBioscience #14061) for 10 minutes at 4° C. Surface staining wasperformed for 20 minutes at 4° C. For intracellular staining, cells wereincubated in Fix/Perm buffer (eBioscience #88-8824) for 20 minutes atroom temperature followed by intracellular cytokine staining withantibody cocktails for 20 minutes at 4° C. After completion of staining,cells were transferred into flow cytometry buffer (PBS with 2% FBS,Penicillin/Streptomycin/Glutamate, EDTA 2 mM) for analysis.

Antibodies used for T cell staining experiments: ICOS FITC (eBioscience#11-9949-80), CD25 AF780 (eBioscience #47-0251-82), CD45.1 AF700(BioLegend #110723), CD8 BV605 (BioLegend #100743), CD4 BV650 (BioLegend#100546), Ki-67 PE-Cy7 (BD Biosciences #561283), CTLA4 PE (BDBiosciences #553720), and FoxP3 PB-e450 (eBiosciences #48-5773-82).

Antibodies used for intracellular cytokine staining experiments: CD3 APC(eBioscience #17-0032-82), NK1.1 APC-AF780 (eBioscience #47-5941-80),CD45.1 AF700 (BioLegend #110723), CD4 BV650 (BioLegend #104729), CD8BV605 (BioLegend #100546), IFN-γ FITC (eBioscience #11-7311-82), IL-17APE-Cy7 (BioLegend #506921), Granzyme-B PE (eBioscience #12-8898-82),FasL PerCP-eFluor710 (eBioscience #46-5911-82), and FoxP3 PB-e450(eBioscience #48-5773-82).

Antibodies used for myeloid cell staining experiments: Ly6G BV785(BioLegend #127645), SiglecF BV785 (BD Biosciences #740956), CD90.2BV785 (BioLegend #10533), B220 BV785 (BioLegend #103246), CD45.1 AF700(BioLegend #110724), CD11b AF780 (eBioscience #47-0112-82), CD206PerCP-Cy5.5 (BioLegend #141716), F4/80 PE (BioLegend #123109), CD11cBV650 (BioLegend #117339), Ly6C BV605 (BioLegend #128035), MHC-IIPB-e450 (eBioscience #48-5321), CD24 PE-Cy7 (BioLegend #101822), CD103FITC (eBioscience #11-1031-82), ADWA11 APC (custom conjugated in ourlaboratory), and CD64 FITC (BioLegend #139316).

Antibodies used for NK cell staining experiments: CD45.1 AF700(BioLegend #110724), CD3 APC (eBioscience #17-0032-82), NK1.1 APC-AF780(eBioscience #47-5941-80), CD314-NKG2D PE (eBioscience #12-5882-82),CD226-DNAM-1 PerCP-Cy5.5 (BioLegend #128814), CD335-NKp46 FITC(eBioscience #11-3351-82), CD107a-LAMP-1 PE-Cy7 (BioLegend #121620), andCD49b eFluor450 (eBioscience #48-5971-82).

For cell stimulation, cells were stimulated prior to cell surface andintracellular staining. Approximately 3×10⁶ cells in 200 μl of C10 mediaper well were incubated in round-bottom 96-well plates overnight in atissue culture incubator in 5% CO2 at 37° C. Stimulation cocktail(Inomycin, PMA, Brefeldin-A, and Monensin 500× stimulation cocktailTombo #TNB4975-UL100) was added to cells which were incubated in tissueculture incubator in 5% CO2 at 37° C. for 4 hours. Cells weretransferred to v-bottom wells for staining as outlined above. Flowcytometry was performed using a BD LSRFortessa™ (BD Biosciences) andanalyzed using FlowJo™ (Tree Star Inc.).

ADWA11 Increases Tumor Infiltration and Enhances Differentiation ofCytotoxic CD8+ T Cells, and Elevates the Ratio of Inflammatory Monocytesto Suppressive Macrophages

The effects of ADWA11 (with or without anti-PD1) on the nature of theimmune infiltrate were also characterized (Thomas et al., Cancer Cell8:369-380, 2005; Wu et al., Cancer Immunol. Res. 2:487-500, 2014).Although treatment with anti-PD1 in this model had no effect on thetotal number of tumor CD8+ T cells, treatment with ADWA11 dramaticallyincreased CD8 T cell infiltration, an effect that was most apparent byimmunostaining excised tumors (FIGS. 19A-19B). ADWA11 also significantlyincreased the percentage of CD8+ T cells that expressed Granzyme B (FIG.19C), whereas treatment with anti-PD1 had no effect. Inflammatorymonocytes also contribute to tumor evasion of host immune responses.Ly6C is expressed on inflammatory monocytes and expression of Ly6C isdiminished in tumor associated macrophages with suppressive propertiesin the tumor microenvironment (Franklin et al., Science 344:921-925,2014; Movahedi et al., Cancer Res. 70:5728-5739). Further classificationof the myeloid population showed that ADWA11 treatment specificallyincreased the accumulation ofCD45+CD11b+CD11c-Ly6G-Ly6C^(high)CD206^(low) inflammatory macrophages(FIG. 19D) (Ostuni et al., Trends Immunol. 36:229-239, 2015; Noy et al.,Immunity 41:49-61, 2014). None of the treatments had significant effectson CD4+ T cell numbers, CD4+ FoxP3+ regulatory T cells, or interferon-γexpression by T cell subsets, at the time points analyzed, and we wereunable to identify significant expression of IL-17 in any T cells subset(data not shown).

For immunostaining, tumors were harvested and fixed in 4%paraformaldehyde at 4° C. overnight. The fixed tumors were immersed in30% sucrose solution at 4° C. overnight and embedded in O.C.T. compound(Tissue Tek® #4583), and cryosectioned at 15 μm. Frozen sections werestained by previously described protocols (Henderson et al., Nat. Med.19:1617-1624, 2013; Rock et al., Proc. Natl. Acad. Sci. USA108:E1475-1483, 2011). In brief, cryosections were permeabilized andblocked with 0.3% Triton X-100 and 3% BSA in PBS. Sections wereincubated with primary antibodies overnight at room temperature, thenwith fluorophore-conjugated primary and secondary antibodies, and thenmounted with Prolong Gold (Invitrogen).

Antibodies used for immunostaining: rat anti-F4/80 (Alexa Fluor647-conjugated, Serotec, clone Cl, 1:100), rabbit anti-phospho-Smad3(Epitomics, 1880-1; 1:100), rat anti-CD8 (Alexa Fluor 488- or594-conjugated, Biolegend, clone 53-6.7, 1:100). Alexa Fluor 488-, 555-,647-conjugated donkey anti-rabbit, and anti-rat (Invitrogen). Confocalmicroscopy was performed on a Zeiss LSM780 and LSMS Pascal microscopes.

All quantifications were done using high-resolution confocal imagesrepresenting a thin (1 airy unit; ˜1 μm) optical section of the sample.Images were analyzed using ImageJ software. Each group contained samplesfrom at least 5 controls, and 5 treated (ADWA11) mice. Four images(fields sized 425.10×425.10 μm) from each tissue section were takenrandomly, using the same confocal settings. Images were placed atidentical thresholds, then the area covered by myofibroblast stains orphospho-Smad3 was calculated.

The Beneficial Synergistic Effects of Combined ADWA11 and Anti-PD1Therapy were Abrogated by CD8+ T Cell Depletion

Since the most dramatic effects of ADWA11 therapy were observed on CD8+T cells, herein it was sought to determine whether these cells drove theanti-tumor effects of ADWA11. CCK168 tumor-bearing mice were depleted ofcytotoxic T cells using an anti-CD-8a (Bio X Cell® BE0004-1 Clone53-6.72) antibody or control antibody at a dose of 10 mg/Kg was injectedintraperitoneally 24 hours prior to each administration of therapeuticdrugs, commencing at day 0. Combined ADWA11 (10 mg/Kg) and anti-PD1 (10mg/Kg) were injected on days 1, 5, and 9. Immunostaining according tothe methods described herein showed effective CD8 depletion (FIG. 21A),which completely abrogated the beneficial effects (e.g., survival andtumor regression) of combination therapy with ADWA11 and anti-PD1 (FIGS.21B-21C).

These data suggest that CD8+ T cells are important in anti-αvb8, e.g.,ADWA11, mediated anti-tumor effects.

pSmad3, a Marker of Active TGFβ Signaling, is Present in CCK168 TumorCells and Cells of the Tumor Microenvironment, and Signaling is BroadlyInhibited by Treatment with ADWA11

Because the best characterized in vivo function of αvβ8 integrin islocal activation of latent TGFβ, it was sought to determine which cellsin untreated tumors showed evidence of TGFβ signaling and whether thissignaling would be inhibited or suppressed by ADWA11 therapy. Toidentify cell types within CCK168 tumors that are actively signalingfrom TGFβ receptors, immunostaining was used with an antibody tophosphorylated SMAD3, a proximal step in TGFβ signaling, as evidencethat cells (CD8+ T cells, F4/80 macrophages and CD11c dendritic cells)were responding to active TGFβ. The CCK168 tumors examined had a richnetwork of macrophages (FIG. 22A) throughout the tumors and only smallnumbers of intercalated DCs. pSMAD3 was readily detected within thetumors, usually in cells adjacent to F4/80+ macrophages, but was notdetected in macrophages themselves. CD8+ T cells were sparse inuntreated mice and were substantially more abundant in tumors from micetreated with AWDA-11. pSMAD3 staining was not observed in these cells.In untreated tumors, high levels of pSMAD3 were seen throughout thetumor microenvironment; however, pSMAD3 staining was broadly inhibitedby treatment with ADWA11 (FIG. 22A). Thus, without wishing to be boundby any particular theory, these data indicate that in some embodiments,αvβ8 activity is essential for TGFβ activation in these tumors, but thatthe effects of TGFβ, when activated by αvβ8, on CD8+ T cellaccumulation, Granzyme B expression and macrophage subset distributionare indirect or occur outside the tumor microenvironment.

Effectorless ADWA11 Inhibits Tumor Growth, Improves Overall Survival andInduces Persistent Anti-Tumor Immunity in CCK168 Squamous Cell CarcinomaModel and CT-26 Carcinomas

Initial studies were performed using a native murine antibody that couldinteract with Fc receptors. It was therefore possible that theanti-tumor effects of ADWA11 could be due toantibody-dependent-cellular-cytotoxicity (ADCC) of tumor cells or tumorinfiltrating macrophages or dendritic cells. To determine the effect ofADCC on ADWA11 activity, a recombinant, Fc “effectorless” version ofADWA11 was generated, termed ADWA11_4mut, by introducing 4 substitutionsinto the IgG1 Fc domain of the mouse antibody to abrogate effectorfunction. These substitutions had previously been shown to completelyabrogate antibody binding to Fc receptors (Alegre et al., J. Immunol.148: 3461-3468, 1992; Hezareh et al., J. Virol. 75: 12161-12168, 2001).The ADWA11_4mut was as effective as the wild type antibody in the CCK168model (FIG. 28). In addition, the ADWA11_4mut antibody was tested forefficacy in two syngeneic tumor models (i.e., the CT26 and EMT6 tumormodels).

CT-26 cells were chosen, in addition to CCK168, since the formercompletely lack detectable αvβ8 expression (FIG. 18B). CT-26 mouse coloncarcinoma cells (ATCC® CRL-2638™) were injected at a dosage of 4×10⁵cells/mouse into the subcutaneous flank of female Balb/c mice (CharlesRiver Labs). Tumors were allowed to grow to 50-100 mm³ in size forinclusion in the study. For these studies, ADWA11_4mut or isotypecontrol 2B8_mIgG_4mut were injected on days 0, 4, 8, 12, anti-PD1antibody (RMP1-14, BioXcell) or isotype control 2A3_rat IgG (BioXcell)were injected intravenously on days 0, 4, 8. All antibodies were dosedat 10 mg/Kg. On Day 5, all mice except the no radiation treatmentcontrol group, were exposed to tumor targeted 5 Gy dose of radiation.Tumor growth was measured twice per week with digital calipers andreported as volume (length×width×width×0.5). For the re-challengeexperiment, on day 51 (post first antibody treatment) mice with acomplete response and naïve mice were implanted on the contralateralflank with 2.5×10⁵ CT26 cells in PBS and tumor growth was monitored asdescribed above.

In both models, the ADWA11_mut4 was effective in driving an anti-tumorresponse (FIG. 28), demonstrating that ADCC function was not requiredfor the ADWA11-mediated anti-tumor effect.

ADWA11 is Efficacious in Multiple Carcinoma Models and Enhances theEffects of Radiation Therapy and Anti-PD-1, Anti-CTLA-4 and 4-1BBTherapy

The efficacy of ADWA11 in other solid tumor models and whether ADWA11could more broadly enhance the beneficial effects of additionalimmunomodulatory therapies were determined. The ability of ADWA11 toenhance the effects of radiation therapy on CT-26 carcinomas was alsoexamined, since this tumor had previously been shown to beradiosensitive, does not express αvβ8 either in vitro or in vivo, andhas been shown to be responsive to a TGFB receptor small moleculeinhibitor (Young et al., PloS One 11:e0157164, 2016). Using radiationdoses that were only minimally effective as therapy, addition of eitherADWA11_4mut or anti-PD-1 significantly increased tumor regression andoverall survival of mice, with 5/9 and 3/10 complete responders,respectively (FIGS. 29A and 29B). Interestingly, the addition ofanti-PD1 to ADWA11 added little additional benefit in this model,providing further evidence that inhibition of αvβ8 can be effective evenin the absence of checkpoint inhibitors. The surviving mice which showedcomplete regression of primary tumors and that received eithermonotherapy or combination therapy were re-challenged on thecontralateral side with the same CT-26 tumor cells at least 51 daysafter initial therapy. Minimal tumor growth was observed with thecontralateral tumors in a few mice. The small tumors that did initiallygrow all subsequently showed complete regression, indicating that insome embodiments, successful treatment with ADWA11 can lead to long-termanti-tumor immunity, as has been previously described for otherimmunomodulators (Ascierto et al., J. Transl. Med. 15:205, 2017) (FIG.29C). None of the primary tumors that were regressed showed re-growth.

ADWA11_4mut is Efficacious in the EMT6 Breast Carcinoma Model andEnhances the Effects of Anti-CTLA-4 and Anti-4-1BB.

The EMT-6 model of breast carcinoma with an immune excluded tumormicroenvironment and low levels of αvβ8 expression was used to examinethe effects of anti-PD-1, anti-CTLA-4 (which has recently been shown towork through a different molecular mechanism than anti-PD1 (Wei et al.,Cell 170:1120-1133, 2017), or an agonist of 4-1BB, a costimulatoryreceptor expressed on CD8+ T cells (Kang et al., Cancer Res. 2017) incombination with ADWA11_4mut.

ADWA11_4mut was also tested in the EMT-6 tumor model. 1×10⁶ EMT6 cells(mouse epithelial mammary carcinoma cell line; ATCC®, CRL2755™) wereinjected into the fourth mammary fad pad of female Balb/c mice (CharlesRiver Labs). Tumors were allowed to grow up to 50-100 mm³ in size. Micewere randomized into antibody treatment groups and operators wereblinded to treatment groups. ADWA11_4mut 10 mg/kg or control 2B8mIgG4mut 10 mg/kg, anti-CTLA4 (9D9 BioXcell) or isotype control E.tenella-mIgG2b 10 mg/kg were injected on days 0, 4 and 8, and anti-4-1BB(MAB9371, R&D systems) 1 mg/kg was injected on days 0 and 4 through anintravenous route. Tumor growth was measured twice per week with digitalcalipers and reported as volume (length×width×width×0.5).

Anti-PD1, anti-CTLA4 or anti-41-BB monotherapy showed significantinitial tumor regression, however only one mouse treated with anti-4-1BBand one treated with anti-PD1 had complete regression (FIG. 30A-D). Incontrast, approximately 70% of mice treated with ADWA11 in combinationwith either anti-PD-1, anti-CTLA4 or anti-4-1BB had complete regression,long-term survival and resistance to EMT6 tumor cell re-challengesuggestive of long-term tumor immunity (FIG. 30E). For the re-challengeexperiment, on day 51 (post first antibody treatment) mice with acomplete response and naïve mice were implanted in the contralateral fatpad with 1×10⁶ EMT-6 cells and tumor growth was monitored as describedabove.

These data demonstrate for the first time that ADWA11 antibodies,including ADWA11 2.4, provide a synergistic therapeutic effect whencombined with an inhibitor of PD-1 or CTLA4 (e.g., an antagonistantibody that binds to PD-1 or CTLA-4 and thereby inhibits the effect ofPD-1 or CTLA-4, respectively) and/or an agonist of 4-1BB (e.g., anagonist antibody that increases the biological activity of 4-1BB). Thesedata suggest that ADWA11 2.4 is a potential human therapeutic that canprovide a synergistic therapeutic anti-tumor response when combined withan inhibitor of, e.g., PD-1 or CTLA4, or an agonist of, e.g., 4-1BB.

Integrin αvβ8 Gene Expression and Staining in Human Tumors

Interrogation of The Cancer Genome Atlas (TCGA) for ITGB8 mRNAexpression revealed that nearly all human tumors, of the thirty tumortypes examined, express detectable robust levels of ITGB8 mRNA, with thehighest levels of expression detected in ovarian and renal cellcarcinomas (FIG. 23A). ITGB8 expression in whole tumor lysates mightreflect expression of αvβ8 on infiltrating immune cells. To test this,multi-panel flow cytometry was performed to assess expression of αvβ8protein in single cells in specimens of freshly harvested anddisaggregated human tumor resections and biopsies. Analysis of two humanovarian carcinomas and two renal cell carcinomas showed substantial αvβ8expressed on CD16+ monocytes, CD14+ tumor associated macrophages andBCDA1+ and BCDA3+ monocyte-derived dendritic cells (FIG. 23B).

Discussion

As will be appreciated by one of ordinary skill in the art, ADWA11 is amonoclonal antibody specific for blocking αvβ8 integrin and is aneffective anti-tumor immunotherapy when used alone. For example, ADWA11demonstrated anti-tumor activity in a CCK168 cutaneous squamous cellcarcinoma. Additionally or alternatively, ADWA11 in combination with animmunomodulator (e.g., anti-PD-1, anti-CTLA-4, and anti-4-1BB) or withradiotherapy demonstrated a powerful and synergistic enhanced anti-tumoractivity in three syngeneic allograft models of epithelial carcinomas onthree different mouse strains backgrounds. In some embodiments,inhibition of αvβ8 increased the number of CD8+ T cells in tumors andtheir cytotoxic differentiation, as assessed by expression of Granzyme Bin these cells. Depletion of CD8+ T cells abrogated the anti-tumoreffects of ADWA11 and anti-PD1 treatment, indicating that in someembodiments, enhanced tumor cell killing by CD8 T cells is critical forthe efficacy of ADWA11. In addition to the effects on CD8+ T cells,ADWA11 increased the number of immunostimulatory monocytes in the tumormicroenvironment (Franklin et al., Science 344:921-925, 2014; Movahediet al., Cancer Res. 70:5728-5739, 2010; Ostuni, Trends Immunol. 36:229-239, 2015; Noy et al., Immunity 41:49-61, 2014). Although there wasno difference in the number of immunosuppressive macrophages, thesemacrophages, along with dendritic cells, expressed high levels ofdetectable αvβ8. Although the first tumor line tested, CCK168 cells,expressed significant surface levels of αvβ8 in vitro, little expressionof αvβ8 was found by flow cytometry on non-hematopoietic cells fromharvested tumors, indicating that in some embodiments, expression islost when these malignant cells form tumors in vivo. Nevertheless, thepossibility that the anti-tumor effects of ADWA11 were due to targetingof the malignant cell directly via ADCC, was excluded by replicating thefindings using a recombinant, effectorless ADWA11 antibody in cells thatdo not express any detectable αvβ8 (i.e., CT-26 colon carcinoma cells).The effectorless antibody was able to suppress tumor growth in threetumor models, CCK168, CT-26 and EMT-6, of which CT-26 has undetectableexrpession of αvβ8. Furthermore, short-term therapy with ADWA11 led tolong-term tumor suppression and resistance to subsequent re-challenge,indicating, in some embodiments, the induction of long-term anti-tumorimmunity. Together, these data indicate that in some embodimentsintegrin αvβ8 blockade causes tumor suppression by blocking αvβ8 oninnate immune cells or T regulatory cells to enhance adaptive immunityto tumors.

TGFβ activation by αvβ8 expressed on dendritic cells, regulatory Tcells, fibroblasts, airway epithelial cells and neuroepithelium, hasbeen shown previously (Travis et al., Nature 449:361-365, 2007; Meltonet al., J. Clin. Invest. 120:4436-4444, 2010; Arnold et al., J.Neurosci. 32(4):1197-1206, 2012; Fenton et al., Mucosal Immunol.10:624-634, 2017; Mu et al., J. Cell Biol. 157:493-507, 2002; Proctor etal., J. Neurosci. 25:9940-9948, 2005; Edwards et al., J. Immunol.193:2843-2849, 2014), but defining the full range of integrin αvβ8expression has been limited by the absence of reliable reagents fortissue staining. In the Examples set forth herein, newly developedantibodies capable of detecting integrin αvβ8 by flow cytometry wereused to demonstrate that tumor associated macrophages and dendriticcells are the major cell types showing high cell surface staining forαvβ8 in murine carcinomas. This indicates that in some embodiments,expression on one or more of these cell types is important forαvβ8-mediated suppression of local anti-tumor immunity.

In the Examples set forth herein, αvβ8 expression on T cells, includingregulatory T cells (Tregs), was not detected.

Activation of TGFβ by integrins, including αvβ8, is tightly spatiallyrestricted, allowing αvβ8-expressing cells to present TGFβ locally tocells they directly contact (Travis et al., Nature 449:361-365, 2007;Munger et al., Cell 96:319-328, 1999). TGFβ has been described tosuppress the activity of effector T cells. As demonstrated in theExamples set forth herein, CD8+ T cells are increased in ADWA11 treatedtumors, are more likely to express Granzyme B, and are important formediating the protective effects of ADWA11. In some embodiments, thesuppressive effects of αvβ8 on innate immune cells is due to directpresentation of active TGFβ to CD8 T cells. However, immunostaining forpSMAD3 did not reveal evidence of TGFβ signaling in CD8+ T cells, butshowed robust signaling in non-hematopoietic cells (e.g. tumor cells).Thus, in some embodiments, tumor cells and some other tumor associatednon-hematopoietic cells (e.g., fibroblasts) are the functionallyimportant cells that respond to TGFβ activated by αvβ8 and suppresslocal tumor immunity.

As described in the Examples set forth herein, αvβ8 integrin is broadlyexpressed on monocytes, macrophages and dendritic cells in multiplemurine and human tumors and is a potent modulator of the anti-tumorimmune response. Monotherapy (e.g., ADWA11) targeting this integrin iseffective in some tumors. Furthermore, efficacy is synergisticallyenhanced by combining inhibition of αvβ8 (e.g., treatment with ADWA11)with either checkpoint inhibitors (e.g., anti-PD1 or anti-CTLA4) or animmune activator (e.g., anti-4-1BB) or by combining αvβ8 monotherapywith radiotherapy. Taken together, these results identify the αvβ8integrin as a novel target for tumor immunotherapy.

Example 15: Further In Vivo Assessment of ADWA11 2.4 Summary of TumorModels for Anti-αvβ8 Evaluation

In vivo efficacy was assessed using syngeneic tumor models inimmunocompetent mice. Efficacy studies were performed with aneffectorless version of the parental mouse hybridoma antibodyADWA11_4mut due to a strong anti-species drug response that limitedexposure with ADWA11 2.4 which is humanized.

TABLE 9 Tumor models for anti-αvβ8 evaluation Tumor Expression of αVTGFp pathway Model microenvironment integrins status EMT6, breast Immuneexcluded Expresses both High TGFp cancer tumor αvβ8 and αvβ6 pathwayactivation microenvironment gene expression model profile CT26, colonPreexisting immune Does not express Low TGFp cancer model αvβ8 or αvβ6pathway activation gene expression profile

Efficacy in the EMT6 Model

The EMT6 tumor model studies were conducted orthotopically (4^(th)mammary fat pad) and treatment with anti-αvβ8 with or without checkpointinhibitors was performed concurrently.

Charles River Balb/c mice (n=10 per group) were implanted with 3×10⁵ or1×10⁶ EMT6 cells/mouse. Treatment was initiated at an average tumorvolume of 50 mm³, or 100 mm³.

The murine parental anti-αvβ8 antibody ADWA11_4mut was dosed at 10 mg/kgonce every four days for a total of three doses in all studies. Anti-PD1antibody (clone RMP1-14, BioXcell) was dosed at 10 mg/kg once every fourdays for a total of three doses, 4-1BB agonist mAb (Clone MAB9371, R&Dsystems) was dosed at 1 mg/kg once every four days for a total of twodoses, and anti-CTLA4 (Clone 9D9, BioXcell) was dosed at 10 mg/kg onceevery four days for a total of three doses.

Anti-αvβ8 monotherapy efficacy of ˜50% tumor growth inhibition (TGI)(Day 10-20) was observed when 3×10⁵ cells were implanted and treatmentwas initiated at 50 mm³. However when 1×10⁶ cells were implanted andtreatment was initiated at 100 mm³ anti-αvβ8 monotherapy TGI was notobserved. The difference in anti-αvβ8 monotherapy TGI between the twostudies is unclear; however, without wishing to be bound by anyparticular theory, the rapid tumor growth rate of this model when 1×10⁶cells were implanted could have limited the response or out competed theanti-tumor response of αvβ8-blockade. Importantly, anti-αvβ8demonstrates a significant synergistic treatment effect with anti-PD1(7/10 complete responses), 4-1BB agonist (5 complete responses), andanti-CTLA4 (6 complete responses), and resulted in an increased survivalas compared to monotherapy and isotype control groups. Therefore, thesedata suggest that ADWA11 antibodies can provide an anti-tumor responsein the EMT-6 tumor model.

Anti-CTLA4 or 4-1BB agonist monotherapy demonstrated significantresponse (˜50% TGI, Day 8-20); however, the response was transient andtumors ultimately outgrew the response. To demonstrate that thecombination treatments resulted in a cellular response to the EMT6tumor, as compared to an anti-angiogenesis or anti-proliferativeresponse, the mice with a complete response were re-challenged on Day 51(after 1^(st) dose) with a second EMT6 tumor implant but withoutadditional drug treatment. EMT6 tumors grew rapidly in naïve mice butwere rapidly cleared in the complete responding mice, indicating in someembodiments, the development of cellular immunity to the tumor cells,e.g., direct tumor cell death, is not mediated by anti-angiogenesis oranti-proliferation.

Quantification of Lymphocyte Abundance in the EMT6 Tumor Model

To investigate the impact of anti-αvβ8 treatment on the EMT6 tumormicroenvironment, the abundance of lymphocyte subsets was quantified byIHC. Briefly, Charles River Balb/c mice (n=10 per group) were implantedwith 1×10⁶ EMT6 cells and treatment was initiated at an average tumorvolume of 100 mm³ (Day 0). Anti-αvβ8 ADWA11 2.4 antibody was dosed at 10mg/kg every three days (e.g., Day 0, Day 3, Day 6, and Day 9) for atotal of four doses, and tumors were harvested on Day 11 (48 hours afterthe 4^(th) dose).

Immunohistochemical (IHC) analysis of the density of CD45 (totallymphocytes and myeloid cells), CD3 (total T cells), CD4 T cells, CD8 Tcells, and Granzyme B (activated CD8 and NK cells) staining demonstratedthat anti-αvβ8 monotherapy increased the abundance of the total CD45,CD4 T cell, CD8 T cells, and very significantly increased the density ofGranzyme B expressing cells (n=10 for each group) (FIG. 15). The IHCdata indicated that in some embodiments, ADWA11 2.4 monotherapy issufficient to change the tumor microenvironment, consistent with theexpected mechanism of action (MOA) of αvβ8.

Efficacy in the CT26 Model

The CT26 model was selected based on the absence of αvβ8 expression,partial response to anti-PD1 therapy, and evidence in the literature fora synergistic TGI of a TGFβ small molecule inhibitor with suboptimaldose of radiation. Radiation therapy (RT) is of particular interest dueto its ability to induce immunological cell death and de novo DC-T cellpriming, where αvβ8 potentially plays an important role in shapingT-cell differentiation and activation. Briefly, Charles River Balb/cmice (n=10 for each group) were subcutaneously implanted with 4e5 cellsand treatment was initiated at an average tumor volume of 100 mm³.

The murine anti-αvβ8 effector null antibody ADWA11_4mut was dosed at 10mg/kg Q4D×4, anti-PD1 (clone RMP1-14, BioXcell) was dosed at 10 mg/kgQ4D×3, and a 5Gy dose of tumor targeted radiation was performed 5 daysafter the first dose of mAbs. Including anti-αvβ8 with RT resulted in asignificant increase in TGI with 5/9 mice showing a complete response.Including anti-PD1 with RT resulted a less significant TGI as comparedto anti-αvβ8, with 3/10 mice demonstrating a complete response. Thetriple combination of RT, anti-αvβ8, and anti-PD1 resulted in a verysignificant TGI with 7/10 mice with a complete response. Similar to theEMT6 study, the mice with a complete response were immune tore-challenge with CT26 cells.

Immunophenotyping of the CT26 Tumor Infiltrating Cell Population

To investigate the impact of anti-αvβ8 treatment on the CT26 tumormicroenvironment the abundance of lymphocyte subsets was quantified byflow cytometry. Briefly, Charles River Balb/c mice (n=6 for each group)subcutaneously implanted with 4e5 cells and treatment was initiated atan average tumor volume of 100 mm³. Anti-αvβ8 ADWA11 2.4 was dosed at 10mg/kg or 1 mg/kg Q3D×3, and tumors were harvested on Day 8 (48 hoursafter the 3^(rd) dose). Tumors were dissociated and the lymphocytepopulation was quantified by CyTOF cytometry. Anti-αvβ8 (10 and 1 mg/kg)increased the abundance of CD8 T cells in the CD3 T cell gate.Additionally, the CD8 cells more frequently expressed Granzyme B, amarker of an activated phenotype.

Summary

Taken together, the EMT6 and CT26 efficacy and PD studies demonstrate:(1) that anti-αvβ8 synergistically increases the response to multiplecheckpoint inhibitors; (2) that anti-αvβ8 efficacy is not dependent uponexpression of αvβ8 by the tumor cell; and (3) that anti-αvβ8 ADWA11 2.4monotherapy is sufficient to increase the abundance of CD8+ GzmB+ Tcellin a tumor microenvironment.

Example 16: ADWA11 2.4 Pharmacokinetics in Human FcRn Transgenic (TG32)Mice

The TG32 mouse model has been shown to be as good as monkey models forpredicting human pharmacokinetics of IgG1 and IgG2 mAbs that exhibitlinear pharmacokinetics. Pharmacokinetics of ADWA11 2.4 was assessed inTG32 mice following a single IV bolus dose administered at 0.1, 0.3, or3 mg/kg. Serum drug concentrations were then measured. As shown in FIG.25A and FIG. 25B, a clear trend of a dose-dependent decrease in antibodyclearance was observed. This is typical for monoclonal antibodies thatinteract with cell surface targets and indicated that the targetreceptor, αvβ8, can act as a clearance mechanism. Consequently, twoseparate pharmacokinetic models were used to describe the linear(typical FcRn mediated antibody clearance mechanism) vs. non-linear(αvβ8-mediated) clearance pathways. For estimation of parameters for thelinear clearance, a two-compartment population pharmacokinetic model wasused to fit the 3 mg/kg data alone (FIG. 25A). For estimation ofpharmacokinetic parameters for the non-linear clearance across multipledoses, a two-compartment saturable model (Michaelis Menten) was used tofit all the data (0.1, 0.3, 3 mg/kg) (FIG. 25B). The estimated linearand non-linear pharmacokinetic parameters are shown in Tables 10 and 11,respectively. The linear pharmacokinetic parameters are within the rangeobserved for typical mAb in TG32 mice.

TABLE 10 Two-compartment pharmacokinetic parameters in TG32 mice (IV at3 mg/kg) Parameter Estimate % CV CL (mL/h/kg) 0.33 9.2 CLD (mL/h/kg) 2.313 V1 (mL/kg) 49 2.4 V2 (mL/kg) 38 9.6 t 1/2 (day) 7.6 CL: clearancefrom central compartment; CLD: inter-compartmental distributionclearance; VI: volume of distribution for the central compartment; V2:volume of distribution for the peripheral compartment; t½, terminalhalf-life calculated based on estimated parameters.

TABLE 11 Two-compartment non-linear pharmacokinetic parameters in TG32mice (IV at 0.1, 0.3, and 3 mg/kg) Parameter Estimate % CV Km (ng/mL)1659 25 Vmax (ng/h/kg) 6596 5 CLD (mL/h/kg) 3.4 20 V1 (mL/kg) 49 4 V2(mL/kg) 48 8 Vmax: maximal rate; Km: substrate concentration to achievehalf maximal rate; CLD inter-compartmental distribution clearance; VI:volume of distribution for the central compartment; V2: volume ofdistribution for the peripheral compartment.

Consistent with the observed target mediated drug disposition (TMDD)based on serum pharmacokinetics, a dose-dependent decrease in drugdistribution into multiple tissues (due to saturating target binding),especially kidney, were also observed in a tissue distribution study.

Example 17: Pharmacokinetics in Non-Human Primates (NHP)

Pharmacokinetics of ADWA11 2.4 was assessed in an NHP exploratorytoxicology study following the first dose of a multiple doseadministration study at 4, 40, or 100 mg/kg. As shown in FIG. 26,exposure of ADWA11 2.4 appears to be linear within this dose range, dueto being above the saturating range for target mediated drug disposition(TMDD). A two-compartment linear model was used to fit the data (FIG.26) and the estimated pharmacokinetics parameters were shown in Table12. Whereas the observed CL and distribution volumes were within rangesfor typical mAbs, the terminal t_(1/2) appears to be on the short end ofthe range. Without wishing to be bound by any particular theory, this islikely due to the terminal phase not well defined as only sampled up to7 days post dose (limit of ETS study design).

TABLE 12 Two-compartment pharmacokinetic parameters in NHP (IV at 4, 40,and 100 mg/kg-first dose) Parameter Estimate % CV CL (mL/h/kg) 0.41 8.7CLD (mL/h/kg) 15.9 25 VI (mL/kg) 35.6 12 V2 (mL/kg) 25.1 25 t½ (day) 4.3CL: clearance from central compartment; CLD: inter-compartmentaldistribution clearance; VI: volume of distribution for the centralcompartment; V2: volume of distribution for the peripheral compartment;t1/2, terminal half-life calculated based on estimated parameters.

Example 18: Pharmacokinetics of Murine Surrogate Antibody asMonotherapy, and when Co-Dosed with Anti-PD-1

Single dose pharmacokinetic data for the parental (mouse IgG) were notavailable, but exposures were measured at up to four time points inseveral efficacy studies and appear to be consistent across thesestudies. Exposures of the parental (mouse IgG) appear to be linearwithin the dose range investigated (1, 3, and 10 mg/kg), consistent withsaturating TMDD as estimated in TG32 mice for the humanized antibody,ADWA11 2.4. A two compartment pharmacokinetic model was fitted toexposure data in the dose-response EMT6 tumor model study, estimatingonly CL while fixing the other parameters at the values reported abovefor humanized antibody in TG32 mouse. The estimated CL of the parental(mouse IgG) in these tumor bearing mice was 0.74 mL/hr/kg.

The exposure of the parental (mouse IgG) appears to be ˜5-50× lowerfollowing repeated co-dosing study with anti-PD-1 antibody, compared tothose dosed with murine surrogate antibody alone or co-dosed withcontrol rat IgG 2A3. This phenomenon was observed in two separatestudies using two different tumor models (CT26 and EMT6). The lowerexposure is possibly due to increased ADA formation in the presence ofanti-PD-1, as has been reported in PD-1 knockout mice (Nishimura H etal., 1998, International Immunol. 10(10): 1563-72). Increased ADAformation following anti-PD-1 antibody (Pembrolizumab) treatment hasalso been reported in the clinic.

Example 19: Human PK/Exposure Prediction

The human pharmacokinetic profile of ADW11 2.4 at doses saturatingtarget mediated drug disposition (TMDD) was predicted by scaling linearpharmacokinetic parameters from TG32 mouse (Table 13). The projectedlinear human pharmacokinetic parameters are given in Table 13. Inaddition, a linear human CL of 0.12 mL/hr/kg was predicted based on themeasured AC-SINS binding (Score=2, Section 2.4), using a platform PBPKmodel. A linear human CL of 0.086 mL/hr/kg was predicted by allometricscaling from NHP. These CL values are within 2× range to the predictionbased on TG32 allometric scaling (0.15 mL/hr/kg), further supportingthat at doses saturating TMDD, ADW11 2.4 will have a pharmacokineticprofile that is typical for monoclonal antibodies.

These results indicate that ADWA11 2.4 is a potential useful humantherapeutic antibody.

TABLE 13 Projected human linear pharmacokinetic parameters for ADWA112.4 Parameter Scaling factor Projected value CL (mL/h/kg) 0.9 0.15 CLD(mL/h/kg) 0.67 0.15 VI (mL/kg) 0.97 39 V2 (mL/kg) 0.93 21 t½ (day) NA 12CL: clearance from central compartment; CLD: inter-compartmentaldistribution clearance; VI: volume of distribution for the centralcompartment; V2: volume of distribution for the peripheral compartment;t1/2, terminal half-life calculated based on estimated parameters.

Additional single-dose ADWA11 (2.4) IV and SC PK and/or TK werecharacterized in male hFcRn TG32 mice (n=4 or 8/dose group and malecynomolgus monkeys (n=1/dose group) and the human pharmacokineticparameters set forth previously herein were revised. More specifically,after single IV dosing (0.1, 0.3, and 3 mg/kg in TG32 mice andcynomolgus monkes), the PK of ADWA11 (2.4) was non-linear in bothspecies with a trend of dose-dependent decrease in CL, consistent withsaturable target-mediated drug disposition. At doses above saturableclearance, the PK of ADWA11 (2.4) is linear and consistent with atypical human IgG1 mAb. The mean PK and TK parameters after single SCdosing at 10 mg/kg, T_(max) was observed at 240 hours post dose, andbioavailability was estimated to be approximately 100% based oncomparison with dose-normalized AUC_(inf) following single IV dosing at3 mg/kg.

ADWA11 (2.4) is expected to be dose-dependent at lower doses than setforth previously elsewhere herein. That is, human PK of ADWA11 (2.4) waspredicted based on allometric scaling of the cynomolgus monkey PK modelthat includes both linear and non-linear clearances. The revisedpredicted human PK parameters are as follows: 36 mL/kg for the volume ofdistribution for the central compartment, 33 mL/kg for volume ofdistribution for the peripheral compartment, 0.12 mL/kg/h for theclearance from central compartment (linear CL), 0.51 mL/kg/h for theinter-compartmental clearance, and the maximum rate of nonlinearelimination of 0.46m/mL/h and a Km of 0.42m/mL for the nonlinearclearance. This model predicts that the nonlinear clearance of ADWA11(2.4) is likely to be saturated above 14 μg/mL plasma concentration witha predicted t_(1/2) of approximately 15 to 17 days.

Example 20: Pharmacokinetics-Pharmacodynamics Relationship andPrediction of Efficacious Human Dose

In the absence of clear exposure-TGI responses in syngeneic mouse tumormodels, the 10 mg/kg dose was considered the efficacious dose forestimation of an efficacious concentration. Using the pharmacokineticmodel for the parental (mouse IgG), the average concentration (C_(avg))was estimated to be 107 μg/mL at day 12 (4 days after the last dose inthe study with Q4D×3 dosing at 10 mg/kg). Similar, though slightlyhigher, C_(avg) values are estimated by calculating AUC using the lineartrapezoid rule from the available exposure data.

As typical antibody pharmacokinetics are expected for ADWA11 2.4 atthese efficacious concentration levels, the two-compartmentpharmacokinetic model with scaled TG32 mouse parameters (Table 13) wereused for human dose prediction (Betts et al. MABS (2018) 1-14). Inaddition, human doses predicted using scaled NHP pharmacokineticparameters differ by only about 20% from those predicted using the TG32mouse. To match the C_(avg), an efficacious human IV dose of 7 mg/kg ispredicted for Q14D×3 dosing, while an IV dose of 12 mg/kg is predictedfor Q28D×3 dosing (FIGS. 27A-27B). The dose may also be tailored toparticular patient populations, clinical indications and/or clinicalsigns and symptoms.

The human PK prediction of ADWA11 2.4 was refined based on allometricscaling from cynomolgus monkeys and outcome of additional preclinicalefficacy studies. Efficacious dose prediction (C_(eff)) was determinedas the plasma concentration that is equal to 10× of the half-maximaleffect on tumor growth inhibition across different studies using theabove 2 mouse tumor models. C_(eff) was estimated to be in the range of10-98 nM across all studies; therefore, a conservative approach was usedto define 100 nM (the high end of the range) as the target C_(eff) Thehuman efficacious dose of ADWA11 2.4 is predicted to be approximately 2mg/kg IV Q14D or 4 mg/kg IV Q28D, which was predicted to provide>IC90tumor growth inhibition coverage at the estimated trough drugconcentration (100 nM). The projected C_(max), C_(ave) and C_(min) atsteady state at the predicted efficacious doses are 84, 44 and 29 μg/mLfollowing 2 mg/kg Q14D (IV), and 132, 45 and 21 μg/mL following 4 mg/kgQ28D (IV), respectively. The predicted human t½ is approximately 15 to17 days at the predicted efficacious dose.

These results support that ADWA11 2.4 is a potential useful humantherapeutic antibody in that it can be dosed in amounts that arecommercially feasible and reasonable to produce.

Example 21: Repeat-Dose Toxicokinetics in CD-1 Mouse

Toxicokinetic and anti-drug antibody evaluations were conducted afterweekly intravenous (IV) or subcutaneous (SC) dosing of ADWA11 2.4 at 10(IV), 100 (SC), or 200 (IV) mg/kg/week for a total of 4 doses to CD-1mice (n=3/sex/dose group) as part of a GLP repeat-dose toxicity study.

There were no quantifiable concentrations of ADWA11 2.4 in samplescollected and analyzed from the vehicle control group at any timepointduring the study. Based on a qualitative review of the data, there wereno consistent sex-related differences in systemic exposure (as assessedby Cmax and AUC168) therefore, group mean toxicokinetic parameters arepresented using combined data from both male and female CD-1 mice (Table18).

Overall mean toxicokinetic parameters for ADWA11 2.4 in CD-I mice. DoseDay Cmax Tmax AUC168 (mg/kg/week)/Route (ug/mL) (hour) (ug:h/mL) 10/IV 1199 0.54 15400 22 615 1.1 46400 100/SC 1 871 72 104000 22 750 18 591000200/IV 1 5020 0.25 258000 22 6510 0.54 267000 AUC₁₆₈ = Area under theconcentration-time curve from time 0 to 168 hours; C_(max) = maximumobserved concentration; T_(max) = time at which Cmax was first observed.

Following IV dosing, systemic exposure increased with increasing dose inan approximately dose-proportional manner on Day 1, and a less thandose-proportional manner on Day 22. The mean accumulation ratios(AUC₁₆₈, Day 22/Day 1) were 3.0, 0.6, and 1.0 for 10 mg/kg/week (IV),100 mg/kg/week (SC) and 200 mg/kg/week (IV), respectively. The lack ofaccumulation following repeat dosing at 100 mg/kg/week (SC) and 200mg/kg/week (IV) could be related to the presence of ADA in animals inthese dose groups.

The overall incidence of ADA induction to ADWA11 2.4 was 28% (5/18animals). The incidence of ADA induction to ADWA11 2.4 was 0% (0/6animals), 67% (4/6 animals) and 17% (1/6 animals) in CD-1 mice dosedwith ADWA11 2.4 at 10 (IV), 100 (SC), or 200 (IV) mg/kg/week,respectively. In general, the exposure in the ADA-positive animals wassimilar or lower compared to the ADA-negative animals.

Example 22: Repeat-Dose Toxicokinetics in Cynomolgus Monkeys

Toxicokinetic and anti-drug antibody evaluations were conducted afterweekly intravenous (IV) or subcutaneous (SC) dosing of ADWA11 2.4 at thedoses of 8 (IV), 100 (SC) and 200 (IV) mg/kg/week for a total of 5 dosesto cynomolgus monkeys as part of a GLP repeat-dose toxicity study.

There were no quantifiable concentrations of ADWA11 2.4 in samplescollected and analyzed from the vehicle control group at any time pointduring the study, or any test article-dosed group prior to dosing onDay 1. There were no apparent sex-related differences in systemicexposures (as assessed by Cmax and AUC168) across dose groups;therefore, group mean TK parameters are discussed and presented usingcombined data from both male and female cynomolgus monkeys (Table 19).

TABLE 19 Overall mean toxicokinetic parameters for ADWA11 2.4 incynomolgus monkeys. Dose Cmax Tmax AUC168 (mg/kg/week)/Route Day (ug/mL)(hour) (ug:h/mL) 8/IV 1 248 0.54 17300 22 394 6.3 44600 100/SC 1 1270 72161000 22 2880 32 393000 200/IV 1 6200 1.1 479000 22 11400 2.7 1100000AUC₁₆₈ = Area under the concentration-time curve from time 0 to 168hours; C_(max) = maximum observed concentration; T_(max) = time at whichCmax was first observed.

Following weekly dosing at 8 (IV), 100 (SC) and 200 (IV) mg/kg, systemicexposure increased approximately dose-proportional on both Day 1 and Day22, with accumulation ratios (Study Day 22/Study Day 1) ranging fromapproximately 2.3 to 2.6.

The overall incidence of ADA induction to ADWA11 2.4 was 39% (7/18animals) across all dose groups. The incidence of ADA induction toADAW11 2.4 was 17% (1/6), 33% (2/6) and 67% (4/6) for animals dosed withADAW11 2.4 at 8 (IV), 100 (SC), or 200 (IV) mg/kg/week dose,respectively. In general, exposure was generally similar in ADA-positiveanimals compared to ADA-negative animals.

Example 23: Non-Clinical Toxicology in Mice and Cynomolgus Monkeys

AWA11 2.4 was administered to mice and cynomolgus monkeys in intravenous(IV) and subcutaneous (SC) studies up to 1 month in duration. The targetorgans identified in these studies included bone, spleen, and clinicalchemistry changes, although the changes were not considered adverse. Theno-adverse-effect levels (NOAELs) in the 1-month studies were 200mg/kg/week IV (C_(max) and AUC168 6510 μg/mL and 267000 μg·h/mL) formice and (C_(max) and AUC168 11,400 μg/mL and 1,100,000 μg·h/mL)cynomolgus monkeys, respectively.

Single-Dose Toxicity

ADWA11 2.4 was tolerated following a single SC dose of 10, 30, or 100mg/kg in female CD-1 mice following by a 2 week observation phase. Therewere no test article-related finding during the study and mean systemicexposure increased with each does in a dose-proportional manner asmeasured by area under the curve (AUC). At 100 mg/kg, the mean C_(max)and AUC₃₃₆ were 795 μg/mL and 148,000 μg*h/mL, respectively.

Repeat-Dose Toxicity

Study 1: In Study 1, female CD-1 mice were administered ADWA11 2.4 by IVbolus at doses of 0, 1, 10, or 100 mg/kg/dose on Days 1, 4, 8, 11, and14. All mice survived the duration of study and all doses weretolerated. At 100 mg/kg/dose, there were test article-relateddifferences in clinical chemistry parameters, compared with controlmean, which included lower glucose (0.75×), and higher phosphorus(1.35×), globulin (1.09×), and blood urea nitrogen (1.18×) without anyassociated microscopic findings or clear dose relationship (glucose andphosphorus). At ≤10 mg/kg/dose, test article-related differences inclinical chemistry parameters included lower glucose (0.77× at 10mg/kg/dose only) and higher phosphorus (1.26×), compared with controlmean. Mean systemic exposure increased with increasing dose in anapproximately dose proportional manner on Day 1 and Day 11. At 100mg/kg/dose, the highest administered dose, the mean C_(max) and AUC48were 4510 μg/mL and 124,000 μg·h/mL on Day 11.

Study 2: In Study 2, cynomolgus monkeys were administered ADWA11 2.4 byIV bolus once weekly at doses of 0, 4, 40, or 100 mg/kg/dose on Days 1,8, and 14. All monkeys survived the duration of the study and all doseswere tolerated. The only test article-related findings were decreases inCD8+ naïve T cells in both the female (0.21× baseline) and male (0.44×baseline) monkeys in the 100 mg/kg/dose group but no changes wereobserved in the 4 or 40 mg/kg/dose groups. Mean systemic exposureincreased with increasing dose in an approximately dose proportionalmanner on Day 1. At 100 mg/kg/dose, the highest administered dose, themean C_(max) and AUC₁₆₈ were 3550 μg/mL and 162,000 μg/mL on Day 1.

Study 3: In Study 3, ADWA11 2.4 was administered to male cynomolgusmonkeys by IV bolus injection on Day 1 at doses of 0.1, 0.3, or 3 mg/kg,or by SC injection at 10 mg/kg. Subsequently, animals which wereadministered 0.1 or 0.3 mg/kg (IV) on Day 1 were also administered 30 or100 mg/kg by SC injection, respectively, on Day 8. All administereddoses/routes of administration were tolerated. There were no testarticle-related in-life observations (i.e., clinical signs, or changesin body weight or food consumption) over a 22- or 29-day observationperiod following dose administration. There was no evidence of erythemaor edema at the injections sites.

Following single IV administration at 0.1, 0.3, and 3 mg/kg on Day 1,the systemic drug exposures increased dose-proportionally between 0.1and 0.3 mg/kg, and more than dose proportionally between 0.3 and 3mg/kg. Following single SC administration at 10 mg/kg on Day 1, or at 30mg/kg and 100 mg/kg on Day 8, the systemic drug exposures increaseddose-proportionally. The SC bioavailability relative to the 3 mg/kg IVdose was 103%, 79%, and 100% at 10, 30, and 100 mg/kg, respectively.Administration of ADWA11 2.4 at 0.1, 0.3, or 3 mg/kg (IV) or 10, 30, or100 mg/kg (SC) to cynomolgus monkeys was tolerated and systemic exposureincreased with dose.

Study 4: In Study 4, ADWA11 2.4 was administered once weekly to male andfemale CD-1 mice by either IV and/or SC doses of 0 (IV and SC), 10 (IV),100 (SC), or 200 (IV) mg/kg/week for 1 month (5 total doses). Testarticle-related findings were limited to nonadverse alterations inclinical chemistry, immunophenotyping parameters in the spleen, andsplenic weights.

All animals survived to scheduled necropsy with the exception of one 100(SC) mg/kg/week male which was found dead on Day 5; however, the deathof this animal was not deemed related to test article. Postmortemautolysis was observed in various organs and a cause of death 200 (IV)mg/kg/week group and included higher globin (1.15× to 1.16×) in bothsexes, lower AG ratio (0.85×) in females, and higher total protein(1.11×) in males compared with concurrent controls. Administration ofthe mAb test article increased the systemic gamma globulin concentrationand likely contributed to these alterations in serum proteins. Becauseof their small magnitude and a lack of associated microscopic ormacroscopic findings, these observations in clinical chemistryparameters were nonadverse. In the spleen, test article-related lowernumbers of CD8+ T cells (0.52×, 0.65×, and 0.60× control mean) infemales and higher percentages of CD4+ T cells expressing the activationmarker CD25 (1.62×, 1.90×, and 1.52× control mean) in males were notedat 10 (IV), 100 (SC), and 200 (IV) mg/kg/week, respectively on Day 30.Mean absolute and relative (to body and brain weights) spleen weightswere lower (0.86× to 0.88× control) in females at 200 (IV) mg/kg/week.There were no correlating macroscopic or microscopic findings for thelower spleen weights. Based on a qualitative review of the data, therewere no consistent sex-related differences in systemic exposure asassessed by C_(max) and AUC₁₆₈. Systemic exposure increased withincreasing IV or SC dose. After repeated dosing, exposures increased at10 mg/kg/week (IV), decreased at 100 (SC) mg/kg/week, and remained thesame at 200 (IV) mg/kg/week. The incidence of anti-drug antibodies (ADA)induction to ADWA11 2.4 was 0% (0/6), 67% (4/6) and 17% (1/6) at 10(IV), 100 (SC), or 200 (IV) mg/kg/week, respectively. There was noevidence of ADWA11 2.4 related microscopic findings in the heart or skinone month after administration of ADWA11 2.4.

Following repeat dose administration, exposures in the ADA-positiveanimals were similar or slightly lower when compared with ADA-negativeanimals. The highest dose administered, 200 (IV) mg/kg/week, wasidentified as the no observed adverse effect level (NOAEL) based on alack of adverse findings at any dose and was associated with a C_(max)of 6,510 μg/mL, and AUC168 of 267,000 μg·h/mL on Day 22.

Study 5: In Study 5, cynomolgus monkeys were administered ADWA11 2.4 asIV and/or SC doses of 0 (IV and SC), 8 (IV), 200 (IV), or 100 (SC)mg/kg/week for 1 month (5 total doses). There were no adverse testarticle-related findings in this study. Nonadverse test article-relatedfindings included unilateral physeal dysplasia in the costochondraljunction in males and alterations in serum proteins (increased globulin(up to 1.29×), total protein (up to 1.10×), and/or decreased albuminglobulin ratio (down to 0.75×) in females at 100 (SC) mg/kg/week andboth sexes at 200 (IV) mg/kg/week. Systemic exposure (as assessed byC_(max) and AUC₁₆₈) was similar in males and females across dose groupsand mean systemic exposure in IV dose groups increased with increasingdose in an approximately dose-proportional manner on Days 1 and 22.Exposures were higher after repeated dosing across groups. There was noevidence of ADWA11 2.4 related microscopic findings in the heart or skinone month after administration of ADWA11 2.4.

The incidence of ADA induction was 17% (1/6 animals), 33% (2/6 animals)and 67% (4/6 animals) for animals dosed with ADWA11 2.4 at 8 (IV), 100(SC), or 200 (IV) mg/kg/week doses, respectively; the incidence of ADAinduction to ADWA11 2.4 was 39% across all dose groups. Serum exposurewas generally similar in ADA-positive animals compared with ADA-negativeanimals. Following once weekly IV or SC administration of ADWA11 2.4,200 (IV) mg/kg/week was identified as the NOAEL in this 1-month toxicitystudy in monkeys based upon a lack of adverse findings in any in-life orpostmortem evaluations. At 200 (IV) mg/kg/week, the mean C_(max) was11,400 μg/mL and the mean AUC₁₆₈ was 1,100,000 μg·h/mL on Day 22.

The NOAELs in the 1-month studies were 200 mg/kg IV for both mice(C_(max) and AUC_(last) 6510 μg/mL and 267000 μg·h/mL) and cynomolgusmonkeys (C_(max) and AUC_(last) 11,400 μg/mL and 1,100,000 μg·h/mL),respectively.

Example 24: In Vitro Complement Protein C1q and FcR Binding

The potential for ADWA11 2.4 to cause complement dependent cytotoxicity(CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) wasinvestigated in in vitro screening assays via binding of C1q and Fcgamma receptors (FcγR), respectively. ADWA11 2.4 did not bind to C1q atthe concentrations tested (up to 30 μg/mL) and therefor is considered tohave a low potential for inducing CDC. ADWA11 2.4 binding to all FcγRstested was similar or lower compared with binding to a negative controlantibody and lower compared with binding to a positive control antibody.Therefore, ADWA11 2.4 is considered to have a low potential to elicitADCC activity.

Example 25: In Vitro Cytokine Release Assay

An in vitro cytokine release assay was performed in whole blood and PBMCformats using samples from 8 human donors. No test-article related TNF,IL-6 or INF-γ release was observed following incubation with ADWA11 2.4.These data are consistent with the lack of changes in serum cytokineprofiles following administration of ADWA11 2.4 in the mouse and monkeystudies described above.

Example 26: Inhibition of αvβ8 Improves the Efficacy of Anti-PD-1Therapy in the MC38 Tumor Model Methods:

In this study, the MC38 murine colon carcinoma cell line was maintainedin Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovineserum, 2 mM glutamine, 100 units/mL penicillin G sodium, 100m/mLstreptomycin sulfate, and 25 μg/mL gentamicin. The cells were culturedin tissue culture flasks in a humidified incubator at 37° C., with anatmosphere of 5% CO₂ and 95% air. On the day of tumor implantation, MC38tumor cells were harvested during exponential growth and resuspended inphosphate buffered saline (PBS). Each mouse received 5×10⁵ cells in a0.1 mL suspension subcutaneously (sc) in the right flank. Tumors weremeasured in two dimensions to monitor growth as the mean volumeapproached 80-120 mm³ range. Volume (V)=½ L×W², and L (length) isdefined as the longest diameter of the tumor and W (width) isperpendicular to L. On Day 1 of the study, animals were sorted intoseven groups (n=10 per group) with group mean tumor volumes of 100 mm³and treated as described below. Tumor measurements were recorded 2-3times per week until tumor volume reached greater than 1200 mm³.

Quantitative PCR (qPCR) analysis of gene expression was performed on 30mg of harvested tumor tissue homogenized in 900 μL of lysis buffersupplied in the RNeasy Plus Mini Kit, using Omin Bead Ruptor. RNA fromhomogenized tumor samples was isolated using the RNeasy Plus Mini Kitand vendor recommended protocols. RNA concentration was quantified usingEpoch BioTek spectrophotometer and resuspended to 200 ng/μL with ddH20.cDNA was synthesized using 2 μg of total RNA and the High-capacity cDNAreverse transcription kit, using vendor recommended protocols. Geneexpression was analyzed using 50 ng of cDNA and gene-specific taqmanprimers, TaqMan Universal Master Mix II, and vendor recommendedprotocols. ViiA7 real-time qPCR system was used for qPCR studies. Thethreshold cycles (CT) for each sample was analyzed using the recommendedcomparative CT method and expression of target genes is reported as foldchange of treatment group compared to isotype control group. Atwo-tailed unpaired Students T-test test was used to compare treatmentgroup to the isotype control group with significance reported at <0.05.

Results:

To investigate the effect of αvβ8-blockade on tumor lymphocyteabundance, tumor tissue was collected Day 12 (antibody treatment on Day1, 5, 9) from mice treated with Isotype control or ADWA11VH05-2/VK01(ADWA 2.4) and analyzed for lymphocyte marker mRNA expressionby quantitative PCR. ADWA11 VH05-2/VK01(ADWA 2.4) treatment increasedthe expression level of CD8a (2.62±0.763) and Granzyme B (5.79±1.55) inthe tumor microenvironment (Fold Change±standard deviation vs Isotypegroup, 10 mg/kg treatment group) (FIG. 31A). These data demonstrate thattreatment with ADWA11 2.4 antibody increases the abundance of activatedlymphocytes, which play a role in tumor regression, in the tumormicroenvironment.

ADWA11 2.4 treatment resulted in an 18% TGI on Day 15 of the study,while anti-PD-1 antibody (RMP1-14) treatment resulted in a 10.3% TGI onDay 16 of the study and no mice reached the end of the study (0%survival at Day 30). By comparison, ADWA11 2.4 in combination withanti-PD-1 resulted in a 35.8% TGI on Day 16 of the study and 60% of micereached the end of the study (% survival at Day 30) (FIG. 31B toppanel).

These results demonstrate that combination therapy of anti-PD1 andanti-αv138, as exemplified by the antibodies used herein, provides anunexpected synergistic antitumor effect. These data indicate thatcombination therapy of the novel anti-αv138 (e.g., ADWA11 2.4)antibodies disclosed herein and anti-PD-1 antibodies well-known in theart, provides a potential novel therapeutic for treatment of tumors.

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications can be made withoutdeparting from the teachings herein and the claimed invention below. Theforegoing examples are provided to better illustrate the disclosedteachings and are not intended to limit the scope of the teachingspresented herein. While the present teachings have been described interms of these exemplary embodiments, the skilled artisan will readilyunderstand that numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety for all purposes. In the event that one ormore of the incorporated literature and similar materials differs fromor contradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1.-31. (canceled)
 32. A method of treating cancer, comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the antibody or antigen-binding fragment thereof of, thatspecifically binds αvβ8 integrin, wherein the antibody orantigen-binding fragment thereof is at least one antibody orantigen-binding fragment thereof selected from the group consisting of:(a) an antibody or antigen-binding fragment thereof, comprising a lightchain complementarity determining region 1 (CDR-L1) comprising the aminoacid sequence of SEQ ID NO:11; a CDR-L2 comprising the amino acidsequence of SEQ ID NO:12; a CDR-L3 comprising the amino acid sequence ofSEQ ID NO:13; a heavy chain CDR1 (CDR-H1) comprising the amino acidsequence of SEQ ID NO:8; a CDR-H2 comprising the amino acid sequence ofSEQ ID NO:9; and a CDR-H3 comprising the amino acid sequence of SEQ IDNO:10; (b) an antibody or antigen-binding fragment thereof, comprising aCDR-L1 comprising the amino acid sequence of SEQ ID NO:17; a CDR-L2comprising the amino acid sequence of SEQ ID NO:18; a CDR-L3 comprisingthe amino acid sequence of SEQ ID NO:19; a CDR-H1 comprising the aminoacid sequence of SEQ ID NO:14; a CDR-H2 comprising the amino acidsequence of SEQ ID NO:15; and a CDR-H3 comprising the amino acidsequence of SEQ ID NO:16; (c) an antibody or antigen-binding fragmentthereof, comprising a variable light (VL) region comprising an aminoacid sequence encoded by the insert of the plasmid deposited with theATCC and having Accession Number PTA-124918, and a variable heavy (VH)region comprising an amino acid sequence encoded by the insert of theplasmid deposited with the ATCC having Accession Number PTA-124917; (d)an antibody or antigen-binding fragment thereof, comprising a VL regioncomprising the amino acid sequence of SEQ ID NO:7, and a VH regioncomprising the amino acid sequence of SEQ ID NO:6; (e) an antibody orantigen-binding fragment thereof, comprising a VL region comprising theamino acid sequence selected from the group consisting of SEQ IDNO:62-66, and a VH region comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO:34-38; (f) an antibody orantigen-binding fragment thereof, comprising a VL region comprising theamino acid sequence selected from the group consisting of SEQ ID NO:47and 92, and a VH region comprising the amino acid sequence selected fromthe group consisting of SEQ ID NO:39 and 88-91; (g) an antibody orantigen-binding fragment thereof, comprising a VL region comprising theamino acid sequence selected from the group consisting of SEQ ID NO:7and 67-69, and a VH region comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO:6 and 93; (h) an antibody orantigen-binding fragment thereof, comprising a VL region comprising theamino acid sequence selected from the group consisting of SEQ ID NO:7,47-69 and 92, and a VH region comprising the amino acid sequenceselected from the group consisting of SEQ ID NO:6, 34-46, 88-91 and 93;(i) an antibody or antigen-binding fragment thereof, comprising a lightchain (LC) region comprising the amino acid sequence of SEQ ID NO:5, anda heavy chain (HC) region comprising the amino acid sequence of SEQ IDNO:2; (j) an antibody or antigen-binding fragment thereof, comprising aLC region comprising the amino acid sequence of SEQ ID NO:5, and a HCregion comprising the amino acid sequence of SEQ ID NO:3; (k) anantibody or antigen-binding fragment thereof, comprising a LC regioncomprising the amino acid sequence of SEQ ID NO:123, and a HC regioncomprising the amino acid sequence of SEQ ID NO:124 or 182; (l) anantibody or antigen-binding fragment thereof, comprising a VL regionencoded by the nucleic acid sequence of SEQ ID NO:186, and a VH regionencoded by the nucleic acid sequence of SEQ ID NO:190; and (m) anantibody or antigen-binding fragment thereof, comprising a LC regionencoded by the nucleic acid sequence of SEQ ID NO:185, and a HC regionencoded by the nucleic acid sequence of SEQ ID NO:189 or
 191. 33. Themethod of claim 32, further administration of a cytotoxic agent, acytostatic agent, a chemotherapeutic agent, a hormone treatment, avaccine, an immunotherapy, surgery, radiation, cryosurgery,thermotherapy, or a combination thereof.
 34. The method of claim 33,wherein the further administration is simultaneous, sequential orseparate from the administration of the therapeutically effective amountof the antibody, or antigen-binding fragment thereof.
 35. The method ofclaim 33, wherein the immunotherapy comprises a modulator of an immunecheckpoint molecule selected from the group consisting of an anti-PD1antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4antibody, a soluble CTLA-4 fusion protein and a combination thereof,wherein the anti-PD-L1 antibody is not avelumab.
 36. The method of claim32, wherein the cancer is selected from the group consisting of squamouscell carcinoma of the head and neck, renal cell carcinoma with clearcell or papillary cell type, ovarian cancer, fallopian tube cancer,primary peritoneal cancer, gastric cancer, gastroesophageal junctioncancer, esophageal cancer, lung squamous cell cancer, pancreatic ductaladenocarcinoma, cholangiocarcinoma, uterine cancer, melanoma, urothelialcarcinoma and combinations thereof. 37.-43. (canceled)
 44. A method oftreating cancer, comprising administering to a subject in need thereof,a therapeutically effective amount of (i) an antibody or antigen-bindingfragment thereof that specifically binds αvβ8 integrin and (ii) amodulator of PD1, PD-L1, or PD-L2.
 45. The method of claim 44, whereinthe cancer is a squamous cell carcinoma.
 46. The method of claim 44,wherein the cancer is breast or colon cancer.
 47. The method of claim44, wherein the modulator is selected from the group consisting of ananti-PD1 antibody, an anti-PD-L1 antibody, and an anti-PD-L2 antibody.